Methods and compositions for promoting a cell-mediated immune response

ABSTRACT

The present invention is directed to a method for promoting or stimulating a cell-mediated immune response to an antigen, by administering a target antigen (such as a protein) with a transport factor that contains a fragment of a bipartite protein exotoxin, but not the corresponding protective antigen. Preferred transport factors include the protective antigen binding domain of lethal factor (LFn) from  B. anthracis , consisting of amino acids 1-255, preferably a fragment of at least 80 amino acids that shows at least 80% homology to LFn, and a fragment of about 105 amino acids from the carboxy portion that does not bind PA. The target antigen can include any molecule for which it would be desirable to elicit a CMI response, including viral antigens and tumor antigens.

CROSS REFERENCE TO RELATED APPLICATION

This Application is a 35 U.S.C. § 371 National Phase Entry Applicationof International Application No. PCT/US2010/038313 filed Jun. 11, 2010,which designates the U.S., and which claims benefit under 35 U.S.C. §119(e) to U.S. Provisional Application 61/186,440, filed, Jun. 12, 2009,the entire contents of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present application is generally directed to compositions fordelivering an exogenous protein to the cytosol of a cell, and methodsand thereof.

BACKGROUND OF THE INVENTION

Vaccines:

Vaccination is the administration of an antigenic material (the vaccine)to produce immunity to a disease. Vaccines can be used to prevent (i.e.prophylactic use) or ameliorate (i.e. therapeutic use) the effects of apathology, for example an infection by a pathogen. Vaccination isconsidered to be the most effective and cost-effective method ofpreventing infectious diseases. The material administered as animmunogen are generally live but weakened or attenuated forms ofpathogens (bacteria or viruses), killed or inactivated forms of thesepathogens, or purified material such as proteins.

The antigenic material (also called an immunogen) stimulates a responsein an animal against the respective pathogen following the initialadministration and also in future encounters with the pathogen, thusproviding protection against infection by the pathogen in futureexposures. Smallpox was the first disease people tried to prevent bypurposely inoculating themselves with other types of infectious agents.For example, the cowpox vaccine was used as an immunization for smallpoxin humans by the British physician Edward Jenner in 1769.

However, not all vaccines and the immunogens in them are equallyeffective in stimulating an effective immune response. For example, poorimmunogenicity of vaccines available against tuberculosis (TB),Streptococcal pneumonia (SP), measles virus Edmoston-Zagreb strain(EZMV), meningococci, hemagglutinin (HA) of influenza viruses, andhepatitis B virus have been reported. Moreover, some vaccines require anextended period of vaccination regime before immunity is successfullyinduced. For example, the primary basic vaccination against Bacillusanthracis requires six doses, three subcutaneous injections in thedeltoid at zero, two, and four weeks, and three vaccinations at six,twelve, and eighteen months followed by annual boosters. For prolongedprotection, annual boosters are required.

One reason accounting for the poor immunogenicity of some vaccines isthe inability of some immunogens to enter the cytosol and the majorhistocompatibility complex pathway in order to stimulate a cell-mediatedimmune response. A number of bacterial toxins contain domains that sharethe ability to gain access to the host cell cytosol, where they canexert their effects. Although each toxin can differ in the mechanism orroute by which it gains entry to the cytosol, the overall effect is thatof a “molecular syringe” that is able to inject the toxic protein intothe cell. Several bacterial toxins, including diphtheria toxin (DT),Pseudomonas exotoxin A (PE), pertussis toxin, and the pertussisadenylate cyclase have been used in attempts to deliver peptide epitopesto the cell cytosol as internal or amino-terminal fusions. These systemsare restricted in their use as potential vaccines because their capacityto deliver larger protein antigens is limited and many individuals havealready been immunized against the carrier toxin.

Although peptides are able to stimulate a cellular immune response,whole protein antigens can be better suited for use in an effectivevaccine for two reasons. First, the epitope that is essential forprotection in one genetic background can prove to be irrelevant in adifferent genetic background. Therefore, it is beneficial for a broadlyapplied T cell vaccine to use the full-length protein from which thevarious relevant epitopes are derived. Second, peptides recognized bycytotoxic T lymphocytes are processed from the whole protein byspecialized degradative machinery, including the proteasome complex. Incertain instances, the processing of the relevant peptide epitopes isdependent on the flanking amino acid sequences. However, flankingresidues are not always important for proper processing. Because itcurrently is not possible to accurately predict which epitopes aredependent on their context for proper processing, it is important todeliver the entire antigen to the cell cytosol for optimal processingand presentation. Therefore, there is a need for new vaccines/immunogensthat are more immunogenic, e. g. immunogens that consist of the wholepolypeptide or larger portions thereof and/or novel strategies forintroducing the vaccines/immunogen into cells to elicit an immuneresponse.

Adjuvants:

The effectiveness of vaccines is often increased by giving them inadjuvants. An effective immune responses against malignancies andagainst several infectious pathogens are mediated by T cells. Inparticular, T helper epitopes are necessary for the induction of hightiters of antigen-specific IgG antibodies. An adjuvant orimmunostimulator can be used to enhance either humoral or cellularimmunity or both. As a result, less recombinant antigen is needed for astandard vaccine or the low-responders respond effectively withoutincreasing the antigen dose.

The primary purpose of an adjuvant is to enhance the immune response toa particular antigen of interest. In the context of antibody productionfor research purposes, adjuvants stimulate the rapid and sustainedproduction of high titers of antibodies with high avidity. This permitsready recovery of antibody for further research in vitro. Adjuvants havethe capability of influencing titer, response duration, isotype, avidityand some properties of cell-mediated immunity. The use of adjuvants isrequired for many antigens which by themselves are weakly immunogenic.

Adjuvants can increase the immune response to a particular antigen ofinterest through three basic mechanisms. The first is to enhance longterm release of the antigen by functioning as a depot. Long termexposure to the antigen should increase the length of time the immunesystem is presented with the antigen for processing as well as theduration of the antibody response. The second is the interaction theadjuvant has with immune cells. Adjuvants can act as non-specificmediators of immune cell function by stimulating or modulating immunecells. Adjuvants can also enhance macrophage phagocytosis after bindingthe antigen as a particulate (a carrier/vehicle function).

The choice of the appropriate adjuvant is exceedingly important fromboth the aspect of the end result (high antibody response) as well asthe immunized animal's welfare. Many of the adjuvants have the capacityto cause inflammation, tissue necrosis and pain in animals, and are thusnot suited for human vaccination.

Typically, selection of an adjuvant is based upon antigencharacteristics (size, net charge and the presence or absence of polargroups), as well as minimizing discomfort. For many years the onlyeffective adjuvant available was complete Freund's adjuvant (CFA). Inthe past, adjuvants have also been selected based upon the species to beimmunized, as some adjuvants will work better than others depending onthe species. However, adjuvant selection remains largely empirical.

Antigens that are easily purified or available in large quantities canbe good choices for starting with the least inflammatory adjuvants forimmunization. Antigens which are difficult to come by (e.g., very smallquantities are available), as well as small molecular weight compoundsor weakly immunogenic antigens are more suitable candidates forcombination with an adjuvant to increase the immune response.

Typically, adjuvants slow antigen release for a more sustained immunestimulation, bind toll-like receptors on macrophages and dendritic cellsto stimulate production of inflammatory cytokines, and/or activateantigen presenting cells to express factors, including IL-10, thatstimulate T cell activation. IL-10 is implicated in the adaptive immuneresponse in humans, in particular, in promoting the development ofTh2-lymphocytes from naïve (Th0) cells. Th2-lymphocytes recognizeantigens presented by B-lymphocytes. Th2 cells, in turn, producecytokines, including interleukins 2, 4, 5, 10, and 13, which promoteantibody production. In addition, the production of IL-4 by Th2 cellsenables the animal to make a quick antibody response that is essentialto resistance to pathogenic antigens.

Collectively these cytokines enable activated B-lymphocytes toproliferate and stimulate activated B-lymphocytes to synthesize andsecrete antibodies, promote the differentiation of B-lymphocytes intoantibody-secreting plasma cells, and enable antibody producing cells toswitch the class of antibodies being produced. Thus, an adjuvant is animportant element of a good immune response to an antigen presented in avaccine.

Complete Freund's Adjuvant (CFA), a mineral oil emulsion adjuvant, was,for many years, the adjuvant of choice because of the ability of CFA toboost antibody production following vaccination. However, CFA, whileimmunogenically potent, frequently produced abscesses, granulomas, andtissue sloughs. In addition, multiple exposures to CFA are known causesevere hypersensitivity reactions, and accidental exposure of personnelto CFA can result in sensitization to the associated antigen. Anothercommon adjuvant frequently used for vaccine antigen delivery is aluminumsalt (“alum”). Most alum adjuvants are generally weaker adjuvants thanemulsion adjuvants, and generally cause only mild inflammatoryreactions. However, alum is best used with strongly immunogenicantigens, and is thus not always appropriate.

Furthermore, in order to generate a CMI response, an antigen must bedelivered to the interior of the cell. Exogenous proteins are poorlytaken up by the cell. Accordingly, the preferred method has been usingprocedures such as viral vectors, liposomes, naked DNA or a similarapproach. However, such approaches have many draw backs. For example,many recombinant viruses generate antigenic reactions themselves, uponrepeated administration. Since standard forms of generating immunereactions typically require an initial injection, referred to as theprime, and subsequent injections, referred to as boosts, to achieve asatisfactory immunity, this can be a serious problem. Moreover, whilemuch attention has been placed on improving the safety of viral vectors,there are always certain risks. For example, many of the targetpopulations, such as those infected with HIV, can have a weakened immunesystem. Thus, certain viral vectors that are perfectly safe in manyindividuals can pose some degree of risk to these individuals.

It would be desirable to use an adjuvant which assist in facilitatingdelivery of an antigen into the interior of a cell in order to generatea CMI response.

SUMMARY OF THE INVENTION

The inventors have previously established that a fragment of the lethalfactor (LF) polypeptide of Bacillus anthracis (B. anthracis) can delivera fused target antigen to the cytosol of an intact cell. In particular,the inventors have previously demonstrated that in the absence of PA, atarget antigen which is covalently attached (i.e. by a covalent bond orfused) to an LF polypeptide such as LFn or a fragment thereof can beused to deliver an antigen to the cytosol of an intact, living cell andelicit a CTL response to the fused antigen. The inventors herein havesurprisingly discovered that it is not necessary for the target antigento be fused to an LF polypeptide to be delivered to the cell cytosoland/or promote a cell-mediated immune response in the absence of PA.Thus, the inventors have now surprisingly discovered that LFpolypeptides, such as LFn and fragments or variants thereof can be usedto promote a cell-mediated immune response in the absence of PA.Accordingly, one aspect of the present invention described hereinrelates to the use of LF polypeptides, such as LFn or fragments orvariants of LFn as an immune adjuvant to promote a cell-mediated immuneresponse without the need for the LFn polypeptide to be covalentlylinked to the antigen. In one aspect, the LF polypeptide useful in themethods and compositions described herein is not physically linked orassociated, or at least not substantially physically associated with thetarget antigen polypeptide. In an alternative aspect, the LF polypeptideis physically associated with the target antigen peptide, e.g. by beingin a non-covalently bound complex with the target antigen polypeptide.

One aspect of the present invention provides compositions for deliveringa target antigen to the cytosol of a cell and uses thereof. Inparticular, one aspect relates to a composition comprising an LFpolypeptide and a target antigen, and methods of using such compositionsto direct an immune response against the antigen, where the compositioncomprises an LF polypeptide and a target antigen which is not covalentlylinked to the LF polypeptide. The LF polypeptide or a fragment thereoffunctions to enhance the efficacy of a vaccine antigen to direct animmune response to the target antigen. In particular, in one aspect,compositions can comprise at least one LF polypeptide, such as forexample, the N-terminal Lethal Factor (LFn) of a bipartite exotoxin suchas B. anthracis, or a fragment or variant thereof, and at least onetarget antigen that is not covalently linked to the LF polypeptide.

Accordingly, one aspect of the present invention relates to acomposition consisting essentially of LF polypeptide, such as forexample, the N-terminal Lethal Factor (LFn) of a bipartate exotoxin suchas B. anthracis and a target antigen, where the LFn is not covalentlylinked to the target antigen and the composition does not comprise aprotective antigen (PA) of B. anthracis.

One aspect of the invention provides a means for eliciting a specificimmune response, in particular a CMI response to a target antigen,whereby the target antigen is delivered to the cytosol of a cell by acomposition consisting essentially of an LF polypeptide, for example,the N-terminal Lethal Factor (LFn) polypeptide or a fragment or variantthereof, and at least one target antigen that is non-linked or notcovalently linked to the LF polypeptide.

In some embodiments, a preferred protein for delivery of antigens to thecytosol of a cell is the N-terminal fragment of the Lethal Factor,herein referred to as “LFn” and corresponds to SEQ ID NO:1. LFn of SEQID NO: 1 is an N-terminal fragment of Lethal Factor (LF) which comprisesa binding region which binds to the protective antigen (PA).

Other aspects of the present invention relate to the use of acomposition comprising an LF polypeptide and a target antigen that isnot covalently linked to the LF polypeptide as described herein. In oneaspect, a composition comprising an LF polypeptide and a target antigenthat is not covalently linked to the LF polypeptide can used in avaccine composition for immunization of a subject against a specifictarget antigen, such as a pathogen (protective or prophylacticvaccination) or for therapeutic treatment for ailments such as cancer,or diseases involving misfolded proteins, gain of function proteins(including for example polyglutamine diseases), aggregated proteins(i.e. disease involving amyloids) as well as specific diseases includingbut not limited to Alzheimer's disease, Creutzfeldt-Jakob disease (CJD),variant Creutzfeldt-Jakob disease (vCJD), ‘Kuru’ or scrapie.

In one embodiment of this aspect, a composition comprising an LFpolypeptide and a target antigen that is not covalently linked to the LFpolypeptide can be used in screening for exposure to pathogens, forexample, in a CMI response assay. An LF polypeptide and non-covalentlylinked target antigen provide a strategy of introducing an antigen intoan intact cell. An LF polypeptide and non-covalently linked targetantigen also provide a strategy to induce an immune response in asubject. In some embodiments, an LF polypeptide and non-covalentlylinked target antigen permit an antigen to enter into a cell, facilitatethe display of the antigen or fragments thereof by MHC molecules so asto induce an immune response in a subject and thereby produce immunityagainst a pathogen having that antigen.

Another aspect of the present invention relates to a method ofintroducing an intracellular pathogen target antigen polypeptide to amammalian cell, the method comprising contacting an LF polypeptide suchas LFn and a target antigen that is not covalently linked to the LFpolypeptide as described herein with a mammalian cell. In someembodiments, an LF polypeptide and a non-covalently linked targetantigen contact the cell concurrently (i.e. simultaneously or at thesame time) or alternatively, a cell can be contacted with an LFpolypeptide and subsequently within a certain time frame anon-covalently linked target antigen, or vice versa. An appropriate timeframe between the contact a cell with an LF polypeptide and anon-covalently linked target antigen can be any time period which allowsthe LF polypeptide to promote transmembrane delivery of thenon-covalently target antigen into the cytosol of an intact cell. Such atime period can be, for example, nanoseconds, milliseconds, seconds oreven minutes.

In one embodiment of this aspect, the LF polypeptide, such as LFnnon-covalently linked to the target antigen promotes transmembranedelivery to the cytosol of an intact cell. In one embodiment, the cellis a mammalian cell in vivo and the method comprises administering an LFpolypeptide and target antigen that is not covalently linked to the LFpolypeptide to the mammal. A target antigen polypeptide normally cannottraverse the plasma membrane and enter an intact cell on its own. Thereare several contributing factors, the size of the polypeptide for one.Proteins in aqueous solutions tend to have their polar amino acidresidues on the outside of the folded structure and the non-polar aminoacid residues on the inside. The lipid bi-layer of the plasma membrane,being non-charged, repels the externally charged protein, preventingtranslocation of the protein across the membrane. Proteins can enter acell by a number of ways, via protein channels which require expenditureof energy or via specific cell surface receptor mediated phagocytosisand/or endocytosis, both of which also require expenditure of energy. AnLF polypeptide, such as LFn can itself traverse the plasma membrane andenter an intact cell on its own. It is known that a target antigen whichis physically linked (i.e. by way of a peptide bond or as fusionprotein) with an LF polypeptide can be translocated into the cytosol ofan intact cell. The inventors herein have surprisingly discovered that atarget antigen polypeptide which is non-covalently linked to LFn canalso be delivered into an intact cell. This method is applicable to anynon-covalently linked target antigen polypeptide, including, but notlimited to., any intracellular pathogen antigen polypeptide. In otherwords, as long as a protein is to be delivered in an intact cell, thismethod can be used to achieve that goal, i. e., by contacting a cellwith a target antigen in the presence of an LF polypeptide such as LFn,the LF polypeptide can promote transmembrane delivery of thenon-covalently linked target antigen to the cytosol of an intact cell.No special knowledge of specific protein channels or specific cellsurface receptor for the target antigen polypeptide is needed. In oneembodiment, an LF polypeptide such as LFn is N-glycosylated. Tointroduce an antigen polypeptide to a mammalian cell, an LF polypeptideand a target antigen that is not covalently linked to the LF polypeptideare simply mixed and contacted with the mammalian cell. In a mammaliansubject, an LF polypeptide such as LFn and a non-covalently linkedtarget antigen can be administered to the subject. Topical and systemicroutes of administration are possible, e. g., parenteral, nasalinhalation, intratracheal, intrathecal, intracranial, intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial,intrasynovial, oral, and intrarectal.

Another aspect of the present invention relates to a method of raising aCMI response to a target antigen polypeptide, the method comprisingadministering to a mammal an LF polypeptide such as LFn and anon-covalently linked target antigen, where the LF polypeptide promotestransmembrane delivery of the non-covalently linked target antigen tothe cytosol of an intact cell. Preferably, an LF polypeptide such as LFnand a non-covalently linked target antigen described as herein areformulated as a vaccine composition for administering to a mammal. Insome aspects, any of the LF polypeptides, such as LFn, andnon-covalently linked target antigens described herein can be used in avaccine composition for immunization of a subject against a specificpathogen. Plotkin and Mortimer (In ‘Vaccines’, 1994, W.B. SaundersCompany; 2nd edition (1994)) provide antigens which can be used tovaccinate animals or humans to induce an immune response specific forparticular pathogens, as well as methods of preparing antigen,determining a suitable dose of antigen, assaying for induction of animmune response, and treating infection by a pathogen (e.g., bacterium,virus, fungus, or parasite). In some embodiments, the target antigen isany pathological target antigen. In some embodiments, an LF polypeptidesuch as an LFn polypeptide can promote the transmembrane delivery of anot covalently linked target antigen which is a whole virus, or anattenuated virus, where the LF polypeptide functions as an adjuvant topromote delivery to the cytosol of the virus, as well as promotinginduction of a CMI response to the whole virus or attenuated virus.

Another aspect of the present invention relates to a vaccine compositioncomprising an LF polypeptide, such as LFn, and a non-covalently linkedtarget antigen, where the LF polypeptide promotes the transmembranedelivery of the non-covalently linked target antigen such as a wholevirus or an attenuated virus, to the cytosol of an intact cell. Anotheraspect of the present invention related to a method of raising a CMIresponse to a target polypeptide, where the method comprisesadministering to a mammal an LF polypeptide such as LFn and anon-covalently linked target antigen, where the LF polypeptide promotestransmembrane delivery of the non-covalently linked target antigen, suchas a whole virus or an attenuated virus, to the cytosol of an intactcell.

Another aspect of the present invention relates to a vaccine compositioncomprising an LF polypeptide such as LFn which is expressed and purifiedfrom insect cells. Also encompassed is a vaccein composion in which boththe LF polypeptide and the target antigen are expressed in insect cells,e.g. using a bacliovirus expression system. In one embodiment, thevaccine composition comprises a plurality of LF polypeptides such as LFnand a plurality of non-covalently linked target antigens that areexpressed and purified from insect cells, wherein the target antigenpolypeptides are different but all are from a single intracellularpathogen. In one embodiment, the plurality of target antigenpolypeptides are all from a single polypeptide from a singleintracellular pathogen. In one embodiment, the vaccine compositioncomprises a plurality of LF polypeptides and a plurality ofnon-covalently linked target antigens. In some embodiments, an LFpolypeptide and plurality of non-covalently linked target antigens areexpressed and purified from insect cells, wherein each target antigenpolypeptide is different but all are from several intracellularpathogens. For example, a vaccine composition raising a cell-mediatedimmune (CMI) response to mumps, measles and rubella viruses can have atleast three different non-covalently linked target antigens, eachspecific to mumps, measles and rubella viruses.

In one aspect, described herein is a composition for promoting a cellmediated immune (CMI) response to a target antigen, the compositioncomprising at least one isolated target antigen and a portion of aLethal Factor (LF) polypeptide lacking LF enzymatic activity, whereinthe portion of an LF polypeptide is not covalently linked to the targetantigen, and wherein the composition does not comprise a protectiveantigen (PA) of an exotoxin bipartite protein.

In one embodiment, the portion of an LF polypeptide comprises at leastthe 60 carboxy-terminal amino acids of SEQ ID NO: 3, or a conservativesubstitution variant thereof that promotes a CMI response to the targetantigen.

In another embodiment, the portion of an LF polypeptide comprises atleast the 80 carboxy-terminal amino acids of SEQ ID NO: 3, or aconservative substitution variant thereof that promotes a CMI responseto the target antigen.

In another embodiment, the portion of an LF polypeptide comprises atleast the 104 carboxy-terminal amino acids of SEQ ID NO: 3, or aconservative substitution variant thereof that promotes a CMI responseto the target antigen.

In another embodiment, the portion of an LF polypeptide comprises theamino acid sequence corresponding to SEQ ID NO: 3 or a conservativesubstitution variant thereof that promotes a CMI response to the targetantigen.

In another embodiment, the portion of an LF polypeptide does not bind PApolypeptide.

In another embodiment, the portion of an LF polypeptide substantiallylacks amino acids 1-33 of SEQ ID NO: 3.

In another embodiment, the portion of an LF polypeptide consists of SEQID NO: 4, or a conservative substitution variant thereof that promotes aCMI response to the target antigen.

In another embodiment, the portion of an LF polypeptide consists of SEQID NO: 5.

In another embodiment, the cell is in vivo or present in an organism.

In another embodiment, the cell is in vitro.

In another embodiment, the composition induces a response by a cellagainst a target antigen, when said cell is contacted with thecomposition in the presence of the target antigen, and in the absence ofan exotoxin protective antigen (PA).

In another embodiment, the target antigen is selected from the groupconsisting of pathogen antigen, a tumor antigen or a endogenousmisfolded protein. In another embodiment, the pathogen antigen isselected from the group consisting of: Hepatitis A, Hepatitis B,Hepatitis C, Avian flu virus, ebola virus, west nile virus, influenzavirus, Herpes Simplex Virus 1, Herpes Simplex Virus2, HIV2, HIV1 andother HIV1 strains.

In another embodiment, the pathogen antigen is not an antigen expressedby B. anthracis.

In another embodiment, the composition optionally comprises at least oneadjuvant. In another embodiment, the adjuvant is selected from a groupcomprising of; complete Freud's Adjuvant, Incomplete Freud's Adjuvant,CM-CSF, QS21, CpG, RIBI Detox, of; IL-2, Ig-IL-2, B7, ICAM, LFS,dextran, polyvinyl pyrrolidones, polysaccharides, starches, polyvinylalcohols, polyacryl amides, polyethylene glycol(PEG), poly(alkylenesoxides), monomethoxy-polyethylene glycol polypropylene glycol, blockcopolymers of polyethylene glycol, polypropylene glycol.

In another aspect, described herein is a method of promotiong acell-mediated immune response to a cell, the method comprisingcontacting said cell with a target antigen in the presence of a portionof a B. anthracis LF polypeptide lacking LF enzymatic activity, whereinthe portion of a B. anthracis LF polypeptide lacking LF enzymaticactivity is not covalently linked to the target antigen, and whereinsaid cell is not contacted with a protective antigen (PA) of an exotoxinbipartite protein, whereby a cell-mediated immune response to the targetantigen is promoted.

In another aspect, described herein is a composition for delivering atarget antigen to a cell, the composition comprising at least one targetantigen and a portion of a B. anthracis LF polypeptide lacking LFenzymatic activity, wherein the portion of a B. anthracis LF polypeptideis not covalently linked to the target antigen, and wherein thecomposition does not comprise a protective antigen of B. anthracisexotoxin bipartite protein.

In one embodiment, the portion of a B. anthracis LF polypeptidecomprises at least the 60 carboxy-terminal amino acids of SEQ ID NO: 3,or a conservative substitution variant thereof that promotestransmembrane delivery.

In another embodiment, the portion of a B. anthracis LF polypeptidecomprises at least the 80 carboxy-terminal amino acids of SEQ ID NO: 3,or a conservative substitution variant thereof that promotestransmembrane delivery.

In another embodiment, the portion of a B. anthracis LF polypeptidecomprises at least the 104 carboxy-terminal amino acids of SEQ ID NO: 3,or a conservative substitution variant thereof that promotestransmembrane delivery.

In another embodiment, the portion of a B. anthracis LF polypeptidecomprises the amino acid sequence corresponding to SEQ ID NO: 3 or aconservative substitution variant thereof that promotes transmembranedelivery.

In another embodiment, the portion of a B. anthracis LF polypeptide doesnot bind B. anthracis PA polypeptide.

In another embodiment, the portion of a B. anthracis LF polypeptidesubstantially lacks amino acids 1-33 of SEQ ID NO: 3.

In another embodiment, the portion of a B. anthracis LF polypeptideconsists of SEQ ID NO: 4, or a conservative substitution variant thereofthat promotes transmembrane delivery.

In another embodiment, the portion of a B. anthracis LF polypeptideconsists of SEQ ID NO: 5.

In another embodiment, the cell is in vivo or present in an organism.

In another embodiment, the cell is in vitro.

In another embodiment, the composition induces a response by a cellagainst a target antigen, when said cell is contacted with thecomposition in the presence of the target antigen, and in the absence ofan exotoxin protective antigen (PA).

In another embodiment, the target antigen is selected from the groupconsisting of pathogen antigen, a tumor antigen or a endogenousmisfolded protein.

In another embodiment, the pathogen antigen is selected from the groupconsisting of: Hepatitis A, Hepatitis B, Hepatitis C, Avian flu virus,ebola virus, west nile virus, influenza virus, Herpes Simplex Virus 1,Herpes Simplex Virus2, HIV2, HIV1 and other HIV1 strains.

In another embodiment, the pathogen antigen is not an antigen expressedby B. anthracis.

In another embodiment, the composition optionally comprises at least oneadjuvant. In another embodiment, the adjuvant is selected from a groupcomprising of; complete Freud's Adjuvant, Incomplete Freud's Adjuvant,CM-CSF, QS21, CpG, RIBI Detox, of; IL-2, Ig-IL-2, B7, ICAM, LFS,dextran, polyvinyl pyrrolidones, polysaccharides, starches, polyvinylalcohols, polyacryl amides, polyethylene glycol(PEG), poly(alkylenesoxides), monomethoxy-polyethylene glycol polypropylene glycol, blockcopolymers of polyethylene glycol, polypropylene glycol.

In another aspect, described hedrein is a method of delivering a targetantigen to the cytosol of a cell, the method comprising contacting saidcell with a target antigen in the presence of a portion of a B.anthracis LF polypeptide lacking LF enzymatic activity, wherein theportion of a B. anthracis LF polypeptide lacking LF enzymatic activityis not covalently linked to the target antigen, and wherein said cell isnot contacted with a protective antigen (PA) of an exotoxin bipartiteprotein, whereby the target antigen is delivered to the cytosol of thecell.

In one embodiment, the delivery of said target antigen induces acell-mediated immune (CMI) response to said target antigen by said cell.

In another embodiment, the portion of an LF polypeptide corresponds toSEQ ID NO: 5 or a functional fragment thereof.

In another embodiment, the portion of a B. anthracis LF polypeptidecomprises at least the 60 carboxy-terminal amino acids of SEQ ID NO: 3,or a conservative substitution variant thereof that promotestransmembrane delivery.

In another embodiment, the portion of a B. anthracis LF polypeptidecomprises at least the 80 carboxy-terminal amino acids of SEQ ID NO: 3,or a conservative substitution variant thereof that promotestransmembrane delivery.

In another embodiment, the portion of a B. anthracis LF polypeptidecomprises at least the 104 carboxy-terminal amino acids of SEQ ID NO: 3,or a conservative substitution variant thereof that promotestransmembrane delivery.

In another embodiment, the portion of a B. anthracis LF polypeptidecomprises the amino acid sequence corresponding to SEQ ID NO: 3 or aconservative substitution variant thereof that promotes transmembranedelivery.

In another embodiment, the portion of a B. anthracis LF polypeptide doesnot bind B. anthracis PA polypeptide.

In another embodiment, the portion of a B. anthracis LF polypeptidesubstantially lacks amino acids 1-33 of SEQ ID NO: 3.

In another embodiment, the portion of a B. anthracis LF polypeptideconsists of SEQ ID NO: 4, or a conservative substitution variant thereofthat promotes transmembrane delivery.

In another embodiment, the portion of a B. anthracis LF polypeptideconsists of SEQ ID NO: 5.

In another embodiment, the cell is in vivo or present in an organism.

In another embodiment, the cell is in vitro.

In another embodiment, the method further comprises administering to thecell at least one other adjuvant, wherein the adjuvant does not compriseSEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the adjuvant isselected from a group comprising of; complete Freud's Adjuvant,Incomplete Freud's Adjuvant, CM-CSF, QS21, CpG, RIBI Detox, of; IL-2,Ig-IL-2, B7, ICAM, LFS, dextran, polyvinyl pyrrolidones,polysaccharides, starches, polyvinyl alcohols, polyacryl amides,polyethylene glycol(PEG), poly(alkylenes oxides),monomethoxy-polyethylene glycol polypropylene glycol, block copolymersof polyethylene glycol, polypropylene glycol.

In another embodiment, the target antigen is selected from the groupconsisting of pathogen antigen, a tumor antigen or a endogenousmisfolded protein.

In another embodiment, the pathogen antigen is selected from the groupconsisting of: Hepatitis A, Hepatitis B, Hepatitis C, Avian flu virus,ebola virus, west nile virus, influenza virus, Herpes Simplex Virus 1,Herpes Simplex Virus2, HIV2, HIV1 and other HIV1 strains.

In another embodiment, the pathogen antigen is not an antigen expressedby B. anthracis.

In another aspect, described herein is the use of a composition asdescribed herein to induce a cell mediated response against a targetantigen by a cell, wherein the cell is contacted with the composition inthe presence of the target antigen, and in the absence of an exotoxinprotective antigen (PA). The composition can be, for example, acomposition comprising at least one isolated target antigen and aportion of a Lethal Factor (LF) polypeptide lacking LF enzymaticactivity, wherein the portion of an LF polypeptide is not covalentlylinked to the target antigen, and wherein the composition does notcomprise a protective antigen (PA) of an exotoxin bipartite protein

In one embodiment, composition further comprises at least one additionalimmune adjuvant. In another embodiment, the immune adjuvant is selectedfrom the group consisting of; Alum, Complete Freud's Adjuvant,Incomplete Freud's Adjuvant, CM-CSF, QS21, CpG, RIBI Detox.

In another embodiment, the immune adjuvant is a cytokine selected fromthe group consisting of; IL-2, Ig-IL-2.

In another embodiment, the immune adjuvant is a co-stimulatory moleculeselected from the group consisting of; B7, ICAM, LFS.

In another embodiment, the immune adjuvant is a non-antigenic polymericsubstance selected from the group consisting of; dextran, polyvinylpyrrolidones, polysaccharides, starches, polyvinyl alcohols, polyacrylamides, polyethylene glycol(PEG), poly(alkylenes oxides),monomethoxy-polyethylene glycol polypropylene glycol, block copolymersof polyethylene glycol, polypropylene glycol.

In another aspect, the method of any of the above uses an LF polypeptideof SEQ ID NO: 3 or SEQ ID NO: 4 that is codon optimized for productionin bacterial cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing depicting the PA-mediated entry of LFn into a cellvia endocytosis, and subsequent presentation by MHC Class I molecules.

FIGS. 2A-E show the amino acid sequence of various Lethal Factor (LF)polypeptides (SEQ ID NO:1). FIG. 2A shows the full length amino acidsequence of LF. FIG. 2B shows the amino acid sequence of the first 288amino acids of LFn (SEQ ID NO:2). FIG. 2C shows the sequence of aminoacids 185-288 of Lethal Factor, sometimes referred to as Fragment 3 (SEQID NO:3). FIG. 2D shows the amino acid sequence of LFn lacking theamino-terminal signal peptide (SEQ ID NO: 4). FIG. 2E shows the aminoacid sequence of one example of a functional fragment of LFn thattransports or increases transport of an antigen across a target cellmembrane. The fragment is a C-terminal fragment (SEQ ID NO: 5).

FIG. 3 shows the domains and secondary structure of the Bacillusanthracis Lethal Factor polypeptide based on the X-ray crystallographydata from Andrew D. Pannifer et. al., (2001). Nature 414, 229-233. TheN-terminal 1-33 amino acid residues are not shown. The consecutivevarying gray-toned regions represent the domains I-IV from N-terminus toC-terminus.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention provides compositions for deliveringa target antigen to the cytosol of a cell and uses thereof. Inparticular, one aspect relates to a composition comprising an LFpolypeptide and a target antigen, and methods of use of using suchcompositions to direct an immune response against an antigen, where thecomposition comprises an LF polypeptide and a non-linked ornon-covalently linked antigen. The LF polypeptide or a fragment thereoffunction to enhance the efficacy of an antigen to direct an immuneresponse to the target antigen. In particular, the compositions of thepresent invention comprise at least one LF polypeptide, for example, theN-terminal Lethal Factor (LFn) of a bipartite exotoxin such as B.anthracis, or a fragment or variant thereof, and at least one non-linkedor non-covalently linked target antigen.

Accordingly, one aspect of the present invention relates to acomposition consisting essentially of LF polypeptide, for example, theN-terminal Lethal Factor (LFn) of a bipartate exotoxin such as B.anthracis, where the LFn is not covalently linked to the target antigenand the composition does not comprise a protective antigen (PA) of B.anthracis.

One aspect of the present invention provides a means for eliciting aspecific immune response, in particular a CMI response to a targetantigen, whereby the target antigen is delivered to the cytosol of acell by a composition consisting essentially of an LF polypeptide, suchas for example, the N-terminal Lethal Factor (LFn) polypeptide or afragment or variant thereof, and a target antigen that is not covalentlylinked to the LF polypeptide.

In some embodiments of this aspect, the composition comprises an LFpolypeptide such as LFn and a non-linked or non-covalently linked targetantigen, meaning that the LF polypeptide such as LFn is not linked tothe target antigen by any covalent bond. In one aspect, the LFpolypeptide is not physically associated with the target antigen in thesubject composition, or, alternatively, not substantially physicallylinked to the target antigen polypeptide. In alternative embodiments, anLF polypeptide can be in a non-covalently linked complex with a targetantigen. For example and as discussed in more detail herein below, insome embodiments, a composition can form an LFn:target antigen complex.In some embodiments, the composition comprises an LFn:target antigencomplex, where an LFn polypeptide, or fragment thereof is associatedwith the target antigen by non-covalent interactions, for example vander Waals forces or other interactions, such as electrostaticinteractions, hydrophilic interactions, hydrophobic interactions and anynon-covalent bond association known by a skilled artisan.

In another embodiment the composition comprises an LFn:target antigencomplex, where an LF polypeptide such as LFn and a non-covalently linkedtarget antigen are in a complex with at least one additional moiety, forexample, a linked polypeptide, where both the target antigen and the LFpolypeptide interacts with the additional moiety. Such interactions canbe any non-covalent bond association known by a skilled artisan,including but not limited to, van der Waals forces, hydrophilicinteractions, hydrophobic interactions and other non-covalentinteractions.

Definitions

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which the invention belongs.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

As used herein, the term “fusion polypeptide” means a protein created byjoining two polypeptide coding sequences together. A fusion polypeptidecan be formed by joining a coding sequence of one polypeptide with acoding sequence of a second polypeptide to form a fusion codingsequence. The fusion coding sequence, when transcribed and translated,expresses a fusion polypeptide.

As used herein, the term “promotes transmembrane delivery” refers to theability of a first polypeptide to facilitate a second polypeptide totraverse the membrane of an intact, living cell. As used in thecompositions and methods described herein, the second protein (i.e.target antigen) is either not linked or is non-covalently linked to thefirst polypeptide (i.e. an LF polypeptide).

As used herein, the term “cytosol” refers to the interior of an intactcell. The “cytosol” comprises the cytoplasm and the organelles inside acell.

As used herein, the term “ an intact cell ” refers to a living cell withan unbroken, uncompromised plasma membrane, and that has a differentialmembrane potential across the membrane, with the inside of the cellbeing negative with respect to the outside of the cell.

The term “adjuvant” as used herein refers to any agent or entity whichincreases the antigenic response by a cell or a subject to a targetantigen.

As used herein, the term “substantially lacks amino acids 1-33” in thecontext of an LF polypeptide described herein refers to an LFpolypeptide that lacks signal peptide activity.

As used herein, the term “intracellular pathogen” refers to a pathogenor components thereof that can reside inside an intact cell.

As used herein, the term “pathogen” refers to an organism or moleculethat causes a disease or disorder in a subject. For example, pathogensinclude but are not limited to viruses, fungi, bacteria, parasites andother infectious organisms or molecules therefrom, as well astaxonomically related macroscopic organisms within the categories algae,fungi, yeast and protozoa or the like.

As used herein, the term “prokaryotic pathogen” refers to a bacterialpathogen.

As used herein, the term “viral pathogen” refers to a virus that causesillness or disease, such as HIV.

As used herein, the term “parasitic pathogen” refers to a microorganismthat is parasitic, residing for an extended period inside a host cell orhost organism, that gains benefits from the host and at the same timecauses illness or disease. A parasitic pathogen can be bacteria,viruses, fungi, and protists.

An “antigen presenting cell” is a cell that expresses the Majorhistocompatibility complex (MHC) molecules and can display foreignantigen complexed with MHC on its surface. Examples of antigendisplaying cells are dendritic cells, macrophages, B cells, fibroblasts(skin), thymic epithelial cells, thyroid epithelial cells, glial cells(brain), pancreatic beta cells, and vascular endothelial cells.

The terms “protective antigen” or “PA” are used interchangeably hereinto refer to part of the B. anthracis exotoxin bipartite protein whichbinds to a mammalian cell's surface by cellular receptors. A “PA,” asthe term is used has its receptor binding site intact and functional.U.S. Pat. Nos. 5,591,631 and 5,677,274 (incorporated by reference intheir entirety) describe PA fusion proteins that target PA to particularcells, such as cancer cells and HIV-infected cells, using as fusionpartners ligands for receptors on the targeted cells.

The term “lethal factor” or “LF” as used herein refers generally to anon-PA polypeptide of the bipartite B. anthracis exotoxin. Wild-type,intact B. anthracis LF polypeptide has the amino acid sequence set outin GenBank Accession Number M29081 (Gene ID No: 143143), whichcorresponds to SEQ ID NO: 1. SEQ ID NO: 1 corresponds to LF with asignal peptide located at residues 1 to 33 at its N-terminus. Statedanother way, immature wild-type LF corresponds to an 809 amino acidprotein, which includes a 33 amino acid signal peptide at theN-terminus. The amino acid sequence of immature wild-type LF (SEQ IDNO: 1) with the signal peptide highlighted in bold is as follows:

(SEQ ID NO: 1)MNIKKEFIKVISMSCLVTAITLSGPVFIPLVQGAGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSLEELKDQRMLSRYEKWEKIKQHYQHWSDSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKELLKRIQIDSSDFLSTEEKEFLKKLQIDIRDSLSEEEKELLNRIQVDSSNPLSEKEKEFLKKLKLDIQPYDINQRLQDTGGLIDSPSINLDVRKQYKRDIQNIDALLHQSIGSTLYNKIYLYENMNINNLTATLGADLVDSTDNTKINRGIFNEFKKNFKYSISSNYMIVDINERPALDNERLKWRIQLSPDTRAGYLENGKLILQRNIGLEIKDVQIIKQSEKEYIRIDAKVVPKSKIDTKIQEAQLNINQEWNKALGLPKYTKLITFNVHNRYASNIVESAYLILNEWKNNIQSDLIKKVTNYLVDGNGRFVFTDITLPNIAEQYTHQDEIYEQVHSKGLYVPESRSILLHGPSKGVELRNDSEGFIHEFGHAVDDYAGYLLDKNQSDLVTNSKKFIDIFKEEGSNLTSYGRTNEAEFFAEAFRLMHSTDHAERLKVQKNAPKTFQFINDQIKFIINS

Cleavage of the immature LF protein results in a mature wild-type LFpolypeptide of 776 amino acids in length. The 776 amino acid polypeptidesequence of mature wild-type LF polypeptide (i.e. lacking the N-terminalsignal peptide) corresponds to SEQ ID NO: 2, as follows:

(SEQ ID NO: 2)AGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSLEELKDQRMLSRYEKWEKIKQHYQHWSDSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKELLKRIQIDSSDFLSTEEKEFLKKLQIDIRDSLSEEEKELLNRIQVDSSNPLSEKEKEFLKKLKLDIQPYDINQRLQDTGGLIDSPSINLDVRKQYKRDIQNIDALLHQSIGSTLYNKIYLYENMNINNLTATLGADLVDSTDNTKINRGIFNEFKKNFKYSISSNYMIVDINERPALDNERLKWRIQLSPDTRAGYLENGKLILQRNIGLEIKDVQIIKQSEKEYIRIDAKVVPKSKIDTKIQEAQLNINQEWNKALGLPKYTKLITFNVHNRYASNIVESAYLILNEWKNNIQSDLIKKVTNYLVDGNGRFVFTDITLPNIAEQYTHQDEIYEQVHSKGLYVPESRSILLHGPSKGVELRNDSEGFIHEFGHAVDDYAGYLLDKNQSDLVTNSKKFIDIFKEEGSNLTSYGRTNEAEFFAEAFRLMHSTDHAERLKVQKNAPKTFQFINDQIKFIINS

The term “LF polypeptide” applies not only to full length, wild-type LF(with or without the signal sequence), but also to fragments thereofthat mediate intracellular delivery of non-covalently linkedpolypeptides to a cell. Also included in the term “LF polypeptide” areconservative substitution variants of LF, including conservativesubstitution variants that mediate such intracellular delivery.

The term “substitution” when referring to a peptide, refers to a changein an amino acid for a different amino-acid moiety. Substitutions can beconservative or non-conservative substitutions, as described furtherherein below.

The term “LFn polypeptide” refers to an N-terminal fragment of B.anthracis LF that does not display zinc metalloproteinase activity,mitogen-activated kinase activity, or both, yet does mediateintracellular or transmembrane delivery of non-covalently linkedpolypeptides. Thus, LFn polypeptides are a subset of LF polypeptides.Each method and/or kit described herein is contemplated to use one ormore LF polypeptides, and not linked or non-covalently linked targetantigen. LFn polypeptides as defined and described herein are preferred.In one aspect, “LFn polypeptide” includes SEQ ID NO: 3, whichcorresponds to a 288 amino acid immature LFn protein; this LFn proteinis “immature” in that it includes a signal peptide located at residues 1to 33 of the N-terminus. Stated another way, immature LFn corresponds toa 288 amino acid protein, which includes a 33 amino acid signal peptideat the N-terminus. Signal peptide cleavage of the immature LFn proteinof SEQ ID NO: 3 results in a mature LFn polypeptide of 255 amino acidsin length. It should be emphasized that, for the purposes of the methodsand compositions described herein, the LF and/or LFn polypeptides caneither include or lack the signal peptide—that is, the presence orabsence of the signal peptide is not expected to influence the activityof LF polypeptides as transmembrane transport facilitators in themethods described herein. The amino acid sequence of immature LFn (SEQID NO: 3) with the signal peptide highlighted in bold is as follows:

(SEQ ID NO: 3)MNIKKEFIKVISMSCLVTAITLSGPVFIPLVQGAGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLS

The polypeptide sequence of a mature LFn polypeptide (which lacks theN-terminal signal peptide) is 255 amino acids in length and correspondsto SEQ ID NO: 4 is as follows:

(SEQ ID NO: 4)AGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLS

The term “functional fragment” as used in the context of a “functionalfragment of LFn” refers to a fragment of an LFn polypeptide thatmediates, effects or facilitates transport of an antigen across anintact, alive cell's membrane. One example of such a fragment of an LFnpolypeptide is a 104 amino acid C-terminal fragment of LFn correspondingto SEQ ID NO: 5 as follows (this sequence is also disclosed as SEQ IDNO: 3 in U.S. patent application Ser. No. 10/473,190, which isincorporated herein by reference):

(SEQ ID NO: 5)GKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLS

The term “LFn polypeptide” as used herein encompasses each of the“immature” LFn and “mature” LFn molecules described herein, as well asfragments, variants (including conservative substitution variants) andderivatives thereof that mediate, effect or facilitate transport of atarget antigen which is not linked, or non-covalently linked, to the LFpolypeptide across the membrane of an intact, living cell. Additionalfragments of LFn polypeptides specifically contemplated for use in themethods, compositions and kits described herein include a fragmentcomprising, or optionally, consisting essentially of the C-terminal 60,80, 90, 100 or 104 amino acids of SEQ ID NO: 3 or a conservativesubstitution variant thereof that mediates, effects or facilitatestransfer of a not-linked or non-covalently linked target antigenpolypeptide across an intact membrane of a living cell, intact cell.

A “fragment” of a target antigen as that term is used herein will be atleast 15 amino acids in length, and can be, for example, at least 16, atleast 17, at least 18, at least 19, at least 20 or at least 25 aminoacids or greater. Thus, in instances where, for example, a panel oftarget antigen fragment-LF polypeptides are prepared, the target antigenfragments will be at least 15 amino acids in length.

The term “Cytotoxic T Lymphocyte” or “CTL” refers to lymphocytes whichinduce apoptosis in targeted cells. CTLs form antigen-specificconjugates with target cells via interaction of TCRs with processedantigen (Ag) on target cell surfaces, resulting in apoptosis of thetargeted cell. Apoptotic bodies are eliminated by macrophages. The term“CTL response” is used to refer to the primary immune response mediatedby CTL cells.

The term “cell mediated immunity” or “CMI” as used herein refers to animmune response that does not involve antibodies or complement butrather involves the activation of macrophages, natural killer cells(NK), antigen-specific cytotoxic T-lymphocytes (T-cells), and therelease of various cytokines in response to a target antigen. Statedanother way, CMI refers to immune cells (such as T cells andlymphocytes) which bind to the surface of other cells that display theantigen (such as antigen presenting cells (APS)) and trigger a response.The response can involve either other lymphocytes and/or any of theother white blood cells (leukocytes) and the release of cytokines.Accordingly, cell-mediated immunity (CMI) is an immune response thatdoes not involve antibodies but rather involves the activation ofmacrophages and NK-cells, the production of antigen-specific cytotoxicT-lymphocytes, and the release of various cytokines in response to anantigen. Cellular immunity protects the body by: (i) activatingantigen-specific cytotoxic T-lymphocytes (CTLs) that are able to destroybody cells displaying epitopes of foreign antigen on their surface, suchas virus-infected cells, cells with intracellular bacteria, and cancercells displaying tumor antigens; (2) activating macrophages and NKcells, enabling them to destroy intracellular pathogens; and (3)stimulating cells to secrete a variety of cytokines that influence thefunction of other cells involved in adaptive immune responses and innateimmune responses. Without wishing to be bound by theory and by way ofbackground, the immune system was separated into two branches: humoralimmunity, for which the protective function of immunization could befound in the humor (cell-free bodily fluid or serum) and cellularimmunity, for which the protective function of immunization wasassociated with cells.

The term “immune cell” as used herein refers to any cell which canrelease a cytokine in response to a direct or indirect antigenicstimulation. Included in the term “immune cells” herein are lympocytes,including natural killer (NK) cells, T-cells (CD4+ and/or CD8+ cells),B-cells, macrophages and monocytes, Th cells; Th1 cells; Th2 cells; Tccells; stromal cells; endothelial cells; leukocytes; dendritic cells;macrophages; mast cells and monocytes and any other cell which iscapable of producing a cytokine molecule in response to direct orindirect antigen stimulation. Typically, an immune cell is a lymphocyte,for example a T-cell lymphocyte.

The term “cytokine” as used herein is used interchangeably with the term“effector molecule,” and refers to a molecule released from an immunecell in response to stimulation with an antigen. Examples of suchcytokines include, but are not limited to; GM-CSF; IL-1α; IL-1β; IL-2;IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL-12; IFN-α; IFN-β; IFN-γ;MIP-1α; MIP-1β; TGF-β; TNFα and TNFβ.

The term “covalently bonded” is meant joined either directly orindirectly (e.g., through a linker) by a chemical bond. Stated anotherway, a covalent bond is a bond between two or more atoms that isprovided by electrons that travel between the atom's nuclei, holdingthem together but keeping them a stable distance apart. Covalent bondsshare electrons between two or more atoms.

The term “complex” as used herein refers to a collection of two or moremolecules, that are physically associated with each other by means otherthan a covalent interaction; for example they can be connected byelectrostatic interactions such as van der Waals forces, etc.

The term “fusion protein” as used herein refers to a recombinant proteinof two or more proteins which are joined by a peptide bond. Fusionproteins can be produced, for example, by a nucleic acid sequenceencoding one protein joined to the nucleic acid encoding another proteinsuch that they constitute a single open-reading frame that can betranslated into a single polypeptide harboring all the intendedproteins.

The term “translocated into a cell” refers to the movement of a moiety,such as the target antigen, and optionally LFn, an LFn homologue ormimetic, or variant thereof from a location the outside the cell, acrossthe plasma membrane to the inside the cell.

The term “transduction” refers to any method whereby a nucleic acid isintroduced into a cell, e.g., by transfection, lipofection,electroporation, biolistics, passive uptake, lipid:nucleic acidcomplexes, viral vector transduction, injection, contacting with nakedDNA, and the like.

The term “in vivo” refers to assays or processes that occur in ananimal.

The term “ex vivo” refers to assays that are performed using a livingcell with an intact membrane that is outside of the body, e.g.,explants, cultured cells, including primary cells and cell lines,transformed cell lines, and extracted tissue or cells, including bloodcells, among others.

The term “in vitro” refers to assays that do not require the presence ofa cell with an intact membrane.

A “cancer cell” refers to a cancerous, pre-cancerous or transformedcell, either in vivo, ex vivo, and in tissue culture, that hasspontaneous or induced phenotypic changes that do not necessarilyinvolve the uptake of new genetic material. Although transformation canarise from infection with a transforming virus and incorporation of newgenomic nucleic acid, or uptake of exogenous nucleic acid, it can alsoarise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene or genes. Transformation/cancer isassociated with, e.g., morphological changes, immortalization of cells,aberrant growth control, foci formation, anchorage independence,proliferation, malignancy, contact inhibition and density limitation ofgrowth, growth factor or serum independence, tumor specific markerslevels, invasiveness, tumor growth or suppression in suitable animalhosts such as nude mice, and the like, in vitro, in vivo, and ex vivo(see also Freshney, Culture of Animal Cells: A Manual of Basic Technique(3rd ed. 1994)).

The term “mammal” is intended to encompass a singular “mammal” andplural “mammals,” and includes, but is not limited to humans; primatessuch as apes, monkeys, orangutans, and chimpanzees; canids such as dogsand wolves; felids such as cats, lions, and tigers; equids such ashorses, donkeys, and zebras, food animals such as cows, pigs, and sheep;ungulates such as deer and giraffes; rodents such as mice, rats,hamsters and guinea pigs; and bears. In some embodiments, a mammal is ahuman. In alternative embodiments, a mammal is not a human.

The term “subject” as used herein refers to any animal in which it isuseful to deliver an exogenous protein to the cytosol of a cell, or todiagnose a CMI response, for example to diagnose if the subject has adisease or condition, or is likely to develop a disease or condition.The subject can be a mammal, for example a human, or can be a wild,domestic, commercial or companion animal. While in one embodiment it iscontemplated that CMI assays are suitable for diagnostic use in humans,it is also applicable to all vertebrates, e.g., mammals, such asnon-human primates, (particularly higher primates), sheep, dog, rodent(e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, andnon-mammals such as chickens, amphibians, reptiles etc. In oneembodiment, the subject is human. In another embodiment, the subject isa wild animal, for example a bird such as for the diagnosis of avianflu. In some embodiments, the subject is an experimental animal oranimal substitute as a disease model. The subject can be a subject inneed of veterinary treatment, including treatment of companion animalssuch as dogs and cats, and domestic animals such as horses, ponies,donkeys, mules, llama, alpaca, pigs, cattle and sheep, or zoo animalssuch as primates, felids, canids, bovids, and ungulates, or livestockanimals such as pigs, cattle and sheep, where the detection of a CMIresponse to a pathogen is useful to prevent a disease and/or to controlthe spread of a disease, for example SIV, STL1, SFV, or in the case oflive-stock, hoof and mouth disease and other such diseases. In someembodiments, a subject is not a human subject.

The term “biological sample” refers to a sample of biological tissue,cells, or fluid that, in a healthy and/or pathological state, containsimmune cells as they are described herein and cells capable ofprocessing and displaying an intracellular polypeptide antigen. Suchsamples include, but are not limited to, whole blood, cultured cells,primary cell preparations, sputum, amniotic fluid, tissue or fine needlebiopsy samples, peritoneal fluid, and pleural fluid, among others. Insome embodiments a biological sample is taken from a human patient, andin alternative embodiments the biological sample is taken from anymammal, such as rodents, animal models of diseases, commercial animals,companion animals, dogs, cats, sheep, cattle, and pigs, etc. Thebiological sample can be pretreated as necessary for storage orpreservation, by dilution in an appropriate buffer solution orconcentrated, if desired. However, the sample must contain living cells.Any of a number of standard aqueous buffer solutions, employing one of avariety of buffers, such as phosphate, Tris, or the like, atphysiological pH can be used. The biological sample can in certaincircumstances be stored prior to use in assays as disclosed herein. Suchstorage can be at +4C or frozen, for example at −20C or -80C, providedsuitable cryopreservation agents are used to maintain cell viabilityonce the cells are thawed.

The term “tissue” refers to a group or layer of similarly specializedcells, which together perform certain special functions. The term“tissue-specific” refers to a source of cells from a specific tissue.The term “tissue” is intended to include, blood, blood preparations suchas plasma and serum, bones, joints, muscles, smooth muscles, and organs.

The term “wild type” refers to the naturally-occurring, normalpolynucleotide sequence encoding a protein, or a portion thereof, orprotein sequence, or portion thereof, respectively, as it normallyexists in vivo. Accordingly, as disclosed herein, the wild type aminoacid sequence for LFn protein corresponds to SEQ ID NO: 3 (with signalpeptide) and/or SEQ ID NO: 4 (without signal peptide), which correspondto an N-terminal fragment of the Lethal Factor (LF) from B. anthracis.

The term “mutant” refers to an organism or cell with any change in itsgenetic material, in particular a change (i.e., deletion, substitution,addition, or alteration) relative to a wild-type polynucleotide sequenceor any change relative to a wild-type protein sequence. The term“variant” is used interchangeably with “mutant”. Although it is oftenassumed that a change in the genetic material results in a change of thefunction of the protein, the terms “mutant” and “variant” refer to achange in the sequence of a wild-type protein regardless of whether thatchange alters the function of the protein (e.g., increases, decreases,imparts a new function), or whether that change has no effect on thefunction of the protein (e.g., the mutation or variation is silent).

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues linked by peptide bonds, and for thepurposes of the claimed invention, have a minimum length of at least 15amino acids. Oligopeptides, oligomer multimers, and the like, typicallyrefer to longer chains of amino acids and are also composed of linearlyarranged amino acids linked by peptide bonds, and whether producedbiologically, recombinantly, or synthetically and whether composed ofnaturally occurring or non-naturally occurring amino acids, are includedwithin this definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also includeco-translational (e.g., signal peptide cleavage) and post-translationalmodifications of the polypeptide, for example, disulfide-bond formation,glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g.,cleavage by furins or metalloproteases), and the like. Furthermore, asused herein, a “polypeptide” refers to a protein that includesmodifications, such as deletions, additions, and substitutions(generally conservative in nature as would be known to a person in theart) to the native sequence, as long as the protein maintains thedesired activity. These modifications can be deliberate, as throughsite-directed mutagenesis, or can be accidental, such as throughmutations of hosts that produce the proteins, or errors due to PCRamplification or other recombinant DNA methods. For the methods, kitsand compositions described herein, the term “peptide” refers to asequence of peptide-linked amino acids containing at least two and lessthan 15 amino acids in length.

It will be appreciated that a protein or polypeptide often containsamino acids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, can be modified in a given polypeptide, eitherby natural processes such as glycosylation and other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Known modifications which can be present inpolypeptides as described herein include, but are not limited to,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa polynucleotide or polynucleotide derivative, covalent attachment of alipid or lipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formulation, gamma-carboxylation, glycation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination.

The terms “homology”, “identity” and “similarity” refer to the degree ofsequence similarity between two peptides or between two optimallyaligned nucleic acid molecules. Homology and identity can each bedetermined by comparing a position in each sequence which can be alignedfor purposes of comparison. For example, it is based upon using astandard homology software in the default position, such as BLAST,version 2.2.14. When an equivalent position in the compared sequences isoccupied by the same base or amino acid, then the molecules areidentical at that position; when the equivalent site occupied by similaramino acid residues (e.g., similar in steric and/or electronic naturesuch as, for example conservative amino acid substitutions), then themolecules can be referred to as homologous (similar) at that position.Expression as a percentage of homology/similarity or identity refers toa function of the number of similar or identical amino acids atpositions shared by the compared sequences, respectively. A sequencewhich is “unrelated” or “non-homologous” shares less than 40% identity,though preferably less than 25% identity with the sequences as disclosedherein.

The term “substantial identity” as used herein denotes a characteristicof a polynucleotide or amino acid sequence, wherein the polynucleotideor amino acid comprises a sequence that has at least 85% sequenceidentity, preferably at least 90% to 95% sequence identity, more usuallyat least 99% sequence identity as compared to a reference sequence overa comparison window of at least 18 nucleotide (6 amino acid) positions,frequently over a window of at least 24-48 nucleotide (8-16 amino acid)positions, wherein the percentage of sequence identity is calculated bycomparing the reference sequence to the sequence which can includedeletions or additions which total 20 percent or less of the referencesequence over the comparison window. The reference sequence can be asubset of a larger sequence. The term “similarity”, when used todescribe a polypeptide, is determined by comparing the amino acidsequence and the conserved amino acid substitutes of one polypeptide tothe sequence of a second polypeptide.

As used herein, the terms “homologous” or “homologues” are usedinterchangeably, and when used to describe a polynucleotide orpolypeptide, indicates that two polynucleotides or polypeptides, ordesignated sequences thereof, when optimally aligned and compared, forexample using BLAST, version 2.2.14 with default parameters for analignment (see herein) are identical, with appropriate nucleotideinsertions or deletions or amino-acid insertions or deletions, in atleast 60% of the nucleotides, usually from about 75% to 99%, and morepreferably at least about 98 to 99% of the nucleotides. The term“homolog” or “homologous” as used herein also refers to homology withrespect to structure and/or function. With respect to sequence homology,sequences are homologs if they are at least 60 at least 70%, at least80%, at least 90%, at least 95% identical, at least 97% identical, or atleast 99% identical. Determination of homologs of the genes or peptidesof the present invention can be easily ascertained by the skilledartisan.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Where necessary or desired, optimal alignment of sequences forcomparison can be conducted, for example, by the local homologyalgorithm of Smith and Waterman (Adv. Appl. Math. 2:482 (1981), which isincorporated by reference herein), by the homology alignment algorithmof Needleman and Wunsch (J. Mol. Biol. 48:443-53 (1970), which isincorporated by reference herein), by the search for similarity methodof Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444-48 (1988),which is incorporated by reference herein), by computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection. (Seegenerally Ausubel et al. (eds.), Current Protocols in Molecular Biology,4th ed., John Wiley and Sons, New York (1999)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show the percent sequence identity. It also plotsa tree or dendogram showing the clustering relationships used to createthe alignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle (J. Mol. Evol. 25:351-60 (1987), which isincorporated by reference herein). The method used is similar to themethod described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53(1989), which is incorporated by reference herein). The program canalign up to 300 sequences, each of a maximum length of 5,000 nucleotidesor amino acids. The multiple alignment procedure begins with thepairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described by Altschul et al. (J. Mol. Biol. 215:403-410 (1990), whichis incorporated by reference herein). (See also Zhang et al., NucleicAcid Res. 26:3986-90 (1998); Altschul et al., Nucleic Acid Res.25:3389-402 (1997), which are incorporated by reference herein).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information internet web site. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.(1990), supra). These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Extension of the wordhits in each direction is halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLAST programuses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix(see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9(1992), which is incorporated by reference herein) alignments (B) of 50,expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci.USA 90:5873-77 (1993), which is incorporated by reference herein). Onemeasure of similarity provided by the BLAST algorithm is the smallestsum probability (P(N)), which provides an indication of the probabilityby which a match between two nucleotide or amino acid sequences wouldoccur by chance. For example, an amino acid sequence is consideredsimilar to a reference amino acid sequence if the smallest sumprobability in a comparison of the test amino acid to the referenceamino acid is less than about 0.1, more typically less than about 0.01,and most typically less than about 0.001.

As used herein, the term “sequence identity” means that twopolynucleotide or amino acid sequences are identical (i.e., on anucleotide-by-nucleotide or residue-by-residue basis) over thecomparison window. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T. C, G, U or I) or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the comparison window (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The term “variant” as used herein refers to a polypeptide or nucleicacid that differs from the naturally occurring polypeptide or nucleicacid by one or more amino acid or nucleic acid deletions, additions,substitutions or side-chain modifications, yet retains one or morespecific functions or biological activities of the naturally occurringmolecule. Amino acid substitutions include alterations in which an aminoacid is replaced with a different naturally-occurring or anon-conventional amino acid residue. Such substitutions can beclassified as “conservative,” in which case an amino acid residuecontained in a polypeptide is replaced with another naturally occurringamino acid of similar character either in relation to polarity, sidechain functionality or size. Substitutions encompassed by variants asdescribed herein can also be “non conservative,” in which an amino acidresidue which is present in a peptide is substituted with an amino acidhaving different properties (e.g., substituting a charged or hydrophobicamino acid with alanine), or alternatively, in which anaturally-occurring amino acid is substituted with a non-conventionalamino acid. Also encompassed within the term “variant,” when used withreference to a polynucleotide or polypeptide, are variations in primary,secondary, or tertiary structure, as compared to a referencepolynucleotide or polypeptide, respectively (e.g., as compared to awild- type polynucleotide or polypeptide). A “variant” of an LFnpolypeptide refers to a molecule substantially similar in structure andfunction to that of a polypeptide of SEQ ID NO: 3, where the function isthe ability to mediate, effect or facilitate transport of a non linkedor non-covalently linked polypeptide across a cell membrane of a livingcell or a living cell present in a subject. In some embodiments, avariant of SEQ ID NO: 3 or SEQ ID NO: 4 is a fragment of SEQ ID NO: 3 or4 as disclosed herein, such as SEQ ID NO: 5.

A molecule is said to be “substantially similar” to another molecule ifboth molecules have substantially similar structures (i.e., they are atleast 50% similar in amino acid sequence as determined by BLASTpalignment set at default parameters) and are substantially similar in atleast one relevant function (here, for example, at least 50% as activein mediating, effecting or facilitating transport of a non-covalentlylinked polypeptide across the membrane of an intact, living cell).Measurement of transmembrane transp;ort can be made, for example, asdescribed by Kushner et al., 2003, Proc. Natl. Acad. Sci. USA, 100(11):6652-6657, which is incorporated herein by reference.

The term “substantially similar,” when used in reference to a variant ofan LF polypeptode, e.g., LFn, or a functional derivative of LFn ascompared to the LFn protein encoded by SEQ ID NO: 3 means that aparticular subject sequence, for example, an LFn fragment or LFn variantor LFn derivative sequence, varies from the sequence of the LFnpolypeptide encoded by SEQ ID NO: 3 by one or more substitutions,deletions, or additions relative to SEQ ID NO: 3, but retains at least50% of the transmembrane transport facilitation activity, and preferablyhigher, e.g., at least 60%, 70%, 80%, 90% or more exhibited by the LFnprotein of SEQ ID NO: 3. (It is acknowledged that LFn does not occurnaturally—reference to a “native” or “natural” LFn sequence is intendedto convey that the sequence is identical to the portion ofnaturally-occurring LF polypeptide designated as LFn herein.) Indetermining polynucleotide sequences, all subject polynucleotidesequences capable of encoding substantially similar amino acid sequencesare considered to be substantially similar to a reference polynucleotidesequence, regardless of differences in codon sequence. A nucleotidesequence is “substantially similar” to a given LFn nucleic acid sequenceif: (a) the nucleotide sequence specifically hybridizes to the codingregions of the native LFn sequence, or (b) the nucleotide sequence iscapable of hybridization to nucleotide sequence of LFn encoded by SEQ IDNO: 1 under moderately stringent conditions and has biological activitysimilar to the native LFn protein; or (c) the nucleotide sequences aredegenerate as a result of the genetic code relative to the nucleotidesequences defined in (a) or (b). Substantially similar proteins willtypically be greater than about 80% similar to the correspondingsequence of the native protein.

Variants can include conservative or non-conservative amino acidchanges, as described below. Polynucleotide changes can result in aminoacid substitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence. Variants can also includeinsertions, deletions or substitutions of amino acids, includinginsertions and substitutions of amino acids and other molecules) that donot normally occur in the peptide sequence that is the basis of thevariant, for example but not limited to insertion of ornithine which donot normally occur in human proteins. “Conservative amino acidsubstitutions” result from replacing one amino acid with another havingsimilar structural and/or chemical properties. Conservative substitutiontables providing functionally similar amino acids are well known in theart. For example, the following six groups each contain amino acids thatare conservative substitutions for one another: 1) Alanine (A), Serine(S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3)Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton,Proteins, W. H. Freeman and Company (1984).)

The choice of conservative amino acids can be selected based on thelocation of the amino acid to be substituted in the polypeptide, forexample if the amino acid is on the exterior of the polypeptide andexposed to solvents, or on the interior and not exposed to solvents.Selection of such conservative amino acid substitutions is within theskill of one of ordinary skill in the art and is described, for exampleby Dordo et al., J. Mol Biol, 1999, 217, 721-739 and Taylor et al., J.Theor. Biol. 119(1986);205-218 and S. French and B. Robson, J. Mol.Evol. 19(1983)171. Accordingly, one can select conservative amino acidsubstitutions suitable for amino acids on the exterior of a protein orpolypeptide (i.e. amino acids exposed to a solvent). These substitutionsinclude, but are not limited to the following: substitution of Y with F,T with S or K, P with A, E with D or Q, N with D or G, R with K, G withN or A, T with S or K, D with N or E, I with L or V, F with Y, S with Tor A, R with K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions suitable for amino acids on the interior of a protein orpolypeptide. For example, one can use suitable conservativesubstitutions for amino acids in the interior of a protein orpolypeptide (i.e. the amino acids are not exposed to a solvent). Forexample, one can use the following conservative substitutions: where Yis substituted with F, T with A or S, I with L or V, W with Y, M with L,N with D, G with A, T with A or S, D with N, I with L or V, F with Y orL, S with A or T and A with S, G, T or V. In some embodiments, LFpolypeptides including non-conservative amino acid substitutions arealso encompassed within the term “variants.” A variant of an LFnpolypeptide, for example a variant of SEQ ID NO: 3 or 4 is meant torefer to any molecule substantially similar in structure (i.e., havingat least 50% homology as determined by BLASTp analysis using defaultparameters) and function (i.e., at least 50% as effective as apolypeptide of SEQ ID NO: 3 in transmembrane transport) to a molecule ofSEQ ID NO: 3 or 4.

As used herein, the term “non-conservative” refers to substituting anamino acid residue for a different amino acid residue that hassubstantially different chemical properties. Non-limiting examples ofnon-conservative substitutions include aspartic acid (D) being replacedwith glycine (G); asparagine (N) being replaced with lysine (K); andalanine (A) being replaced with arginine (R).

The term “derivative” as used herein refers to peptides which have beenchemically modified, for example by ubiquitination, labeling, pegylation(derivatization with polyethylene glycol) or addition of othermolecules. A molecule is also a “derivative” of another molecule when itcontains additional chemical moieties not normally a part of themolecule. Such moieties can improve the molecule's solubility,absorption, biological half life, etc. The moieties can alternativelydecrease the toxicity of the molecule, or eliminate or attenuate anundesirable side effect of the molecule, etc. Moieties capable ofmediating such effects are disclosed in Remington's PharmaceuticalSciences, 18th edition, A. R. Gennaro, Ed., MackPubl., Easton, Pa.(1990).

The term “functional” when used in conjunction with “derivative” or“variant” refers to a protein molecule which possess a biologicalactivity that is substantially similar to a biological activity of theentity or molecule of which it is a derivative or variant. By“substantially similar” in this context is meant that the biologicalactivity, e.g., transmembrane transport of associated polypeptides is atleast 50% as active as a reference, e.g., a corresponding wild-typepolypeptide, and preferably at least 60% as active, 70% as active, 80%as active, 90% as active, 95% as active, 100% as active or even higher(i.e., the variant or derivative has greater activity than thewild-type), e.g., 110% as active, 120% as active, or more.

“Insertions” or “deletions,” as the terms are used herein, are typicallyin the range of about 1 to 5 amino acids. Where necessary, the variationpermitted in view of maintaining function can be experimentallydetermined by producing the polypeptide synthetically whilesystematically making insertions, deletions, or substitutions ofnucleotides in the sequence using recombinant DNA techniques.

The term “specifically binds” refers to binding with a Kd of 10micromolar or less, preferably 1 micromolar or less, more preferably 100nM or less, 10 nM or less, or 1 nM or less.

By “substantially pure” is meant a nucleic acid, polypeptide, or othermolecule that has been separated from the components that naturallyaccompany it. Typically, a polypeptide is substantially pure when it isat least about 60%, or at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, or even at least about 99%, by weight,free from the proteins and naturally occurring organic molecules withwhich it is naturally associated. A substantially pure polypeptide canbe obtained, for example, by extraction from a natural source, byexpression of a recombinant nucleic acid in a cell that does notnormally express that protein, or by chemical synthesis.

The terms “reduced” or “reduce” or “decrease” as used herein generallymean a decrease by a statistically significant amount relative to areference. However, for avoidance of doubt, “reduced” or “decreased”mean a statistically significant decrease of at least 10% as compared toa reference level, for example a decrease by at least 20%, at least 30%,at least 40%, at least t 50%, or least 60%, or least 70%, or least 80%,at least 90% or more, up to and including a 100% decrease (i.e. absentlevel as compared to a reference sample), or any decrease between10-100% as compared to a reference level, for example a control sample,such as a negative control which has a absence of an agent or absence ofa condition (such as absence of a LF polypeptide).

The term “low” as used herein generally means lower by a staticallysignificant amount; for the avoidance of doubt, “low” means astatistically significant value at least 10% lower than a referencelevel, for example a value at least 20% lower than a reference level, atleast 30% lower than a reference level, at least 40% lower than areference level, at least 50% lower than a reference level, at least 60%lower than a reference level, at least 70% lower than a reference level,at least 80% lower than a reference level, at least 90% lower than areference level, up to and including 100% lower than a reference level(i.e. absent level as compared to a reference sample), where thereference level can be a control sample, such as a negative controlsample, such as a sample in the absence of an agent or absence of acondition (such as absence of a LF polypeptide).

The terms “increased” or “increase” as used herein generally mean anincrease by a statically significant amount; for the avoidance of doubt,“increased” means a statistically significant increase of at least 10%as compared to a reference level, including an increase of at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100% or more, including, for exampleat least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 10-fold increase or greater as compared to a reference level, suchas a control condition, such as a negative control or in the absence ofa condition (such as absence of an LF polypeptide).

The term “high” as used herein generally means a higher by a staticallysignificant amount relative to a reference; for the avoidance of doubt,“high” means a statistically significant value at least 10% higher thana reference level, for example at least 20% higher, at least 30% higher,at least 40% higher, at least 50% higher, at least 60% higher, at least70% higher, at least 80% higher, at least 90% higher, at least 100%higher, at least 2-fold higher, at least 3-fold higher, at least 4-foldhigher, at least 5-fold higher, at least 10-fold higher or more, ascompared to a reference level, such as a control condition, such as anegative control or in the absence of a condition (such as absence of anLF polypeptide).

The term “recombinant” as used herein to describe a nucleic acidmolecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic,and/or synthetic origin, which, by virtue of its origin or manipulation,is not associated with all or a portion of the polynucleotide sequenceswith which it is associated in nature. The term recombinant as used withrespect to a protein or polypeptide, means a polypeptide produced byexpression from a recombinant polynucleotide. The term recombinant asused with respect to a host cell means a host cell into which arecombinant polynucleotide has been introduced. Recombinant is also usedherein to refer to, with reference to material (e.g., a cell, a nucleicacid, a protein, or a vector) that the material has been modified by theintroduction of a heterologous material (e.g., a cell, a nucleic acid, aprotein, or a vector).

The term “vectors” refers to a nucleic acid molecule capable oftransporting or mediating expression of heterologous nucleic acid towhich it has been linked; a plasmid is a species of the genusencompassed by the term “vector”. The term “vector” typically refers toa nucleic acid sequence containing an origin of replication and otherentities necessary for replication and/or maintenance in a host cell.Vectors capable of directing the expression of genes and/or nucleic acidsequence to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility areoften in the form of “plasmids” which refer to circular double strandedDNA loops which, in their vector form are not bound to the chromosome,and typically comprise entities for stable or transient expression orthe encoded DNA. Other expression vectors that can be used in themethods as disclosed herein include, but are not limited to, plasmids,episomes, bacterial artificial chromosomes, yeast artificialchromosomes, bacteriophages or viral vectors, and such vectors canintegrate into the host's genome or replicate autonomously in theparticular cell. A vector can be a DNA or RNA vector. Other forms ofexpression vectors known by those skilled in the art which serve theequivalent functions can also be used, for example self replicatingextrachromosomal vectors or vectors which integrates into a host genome.Preferred vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked.

As used herein, the terms “treat” or “treatment” or “treating” refer toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow the development of the disease.Without wishing to be limited by examples, if the disease is cancer, theslowing of the development of a tumor, the spread of cancer, or reducingat least one effect or symptom of a condition, disease or disorderassociated with inappropriate proliferation or a cell mass, for examplecancer would be considered a treatment. Where the disease is, forexample, an infection, such as an HIV infection, a decrease of virustiter, or increase in white blood cells, or an improvement, orattenuating the decline in a symptom of Auto-Immune Disease syndrome(AIDS) is considered treatment. Treatment is generally “effective” ifone or more symptoms or clinical markers are reduced as that term isdefined herein. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation or at least slowing of progress or worsening of symptoms thatwould be expected in the absence of treatment. Beneficial or desiredclinical results include, but are not limited to, alleviation of one ormore symptom(s), diminishment of extent of disease, stabilized (i.e.,not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already diagnosed with cancer, as well as thoselikely to develop secondary tumors due to metastasis.

The term “effective amount” as used herein refers to the amount oftherapeutic agent or pharmaceutical composition to alleviate at leastone or more symptom of a targeted disease or disorder, and relates to asufficient amount of pharmacological composition to provide the desiredeffect. Alternatively, the term refers to the amount necessary todeliver an exogenous protein or polypeptide to the cytosol of a cell.The phrase “therapeutically effective amount” as used herein, e.g., ofany composition as disclosed herein means a sufficient amount of thecomposition to treat a disorder, at a reasonable benefit/risk ratioapplicable to any medical treatment. The term “therapeutically effectiveamount” therefore refers to an amount of the composition as disclosedherein that is sufficient to effect a therapeutically orprophylactically significant reduction in a symptom or clinical markerassociated with a disease.

A therapeutically or prophylatically significant reduction in a symptomis, e.g. at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, up to and including atleast about 100% or more in a measured parameter as compared to acontrol or non-treated subject. Measured or measurable parametersinclude clinically detectable markers of disease, for example, elevatedor depressed levels of a biological marker, as well as parametersrelated to a clinically accepted scale of symptoms or markers for adisease or disorder. It will be understood, however, that the totaldaily usage of the compositions and formulations as disclosed hereinwill be decided by the attending physician within the scope of soundmedical judgment. The exact amount required will vary depending onfactors such as the type of disease being treated.

With reference to the treatment of a subject with a cancer, the term“therapeutically effective amount” refers to the amount that is safe andsufficient to prevent or delay the development and further growth of atumor or the spread of metastases in cancer patients. The amount canthus cure or cause the cancer to go into remission, slow the course ofcancer progression, slow or inhibit tumor growth, slow or inhibit tumormetastasis, slow or inhibit the establishment of secondary tumors atmetastatic sites, or inhibit the formation of new tumor metastases. Theeffective amount for the treatment of cancer depends on the tumor to betreated, the severity of the tumor, the drug resistance level of thetumor, the species being treated, the age and general condition of thesubject, the mode of administration and so forth. Thus, it is notreasonable to specify the exact “effective amount”. However, for anygiven case, an appropriate “effective amount” can be determined by oneof ordinary skill in the art using only routine experimentation. Theefficacy of treatment can be judged by an ordinarily skilledpractitioner. For example, efficacy can be assessed in animal models ofcancer and tumor, for example treatment of a rodent having anexperimental cancer, and any treatment or administration of thecompositions or formulations that leads to a decrease of at least onesymptom of the cancer, for example a reduction in the size of the tumoror a slowing or cessation of the rate of growth of the tumor indicateseffective treatment. In embodiments where the compositions are used forthe treatment of cancer, the efficacy of the composition can be judgedusing an experimental animal model of cancer, e.g., mice or rats, or forexample, transplantation of tumor cells, e.g. xenograft animal cancermodels, or an animal model which has been genetically modified todevelop cancer. Further, in some embodiments an experimental model couldbe an in vitro model, such as organ culture, cells or cell lines. Whenusing an experimental animal model, efficacy of treatment is evidencedwhen a reduction in a symptom of the cancer, for example a reduction inthe size of the tumor or when a slowing or cessation of the rate ofgrowth of the tumor occurs earlier in treated, versus untreated animals.By “earlier” is meant that a decrease, for example in the size of thetumor occurs at least 5% earlier, but preferably more, e.g., one dayearlier, two days earlier, 3 days earlier, or more.

As used herein, the terms “administering,” and “introducing” are usedinterchangeably and refer to the placement of agents as disclosed hereininto a subject by a method or route which results in delivering of suchagent(s) at a desired site. The compounds can be administered by anyappropriate route which results in an effective treatment in thesubject.

The terms “composition” or “pharmaceutical composition” usedinterchangeably herein refer to compositions or formulations thatcomprise an LF polypeptide and at least one target antigen that is notcovalently linked to the LF polypeptide. In some embodiments, apharmaceutical composition can also optionally comprise an excipient,such as a pharmaceutically acceptable carrier that is conventional inthe art and that is suitable for administration to mammals, andpreferably humans or human cells. Such compositions can be specificallyformulated for administration via one or more of a number of routes,including but not limited to, oral, ocular parenteral, intravenous,intraarterial, subcutaneous, intranasal, sublingual, intraspinal,intracerebroventricular, and the like. In addition, compositions fortopical (e.g., oral mucosa, respiratory mucosa) and/or oraladministration can form solutions, suspensions, tablets, pills,capsules, sustained-release formulations, oral rinses, or powders, asknown in the art are described herein. The compositions also can includestabilizers and preservatives. For examples of carriers, stabilizers andadjuvants, University of the Sciences in Philadelphia (2005) Remington:The Science and Practice of Pharmacy with Facts and Comparisons, 21stEd.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration therapeutic compositions other than directly into a tumorsuch that it enters the animal's system and, thus, is subject tometabolism and other like processes.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. The phrase“pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in maintaining the activity or solubility of, orcarrying or transporting the subject agents from one organ, or portionof the body, to another organ, or portion of the body. In addition tobeing “pharmaceutically acceptable” as that term is defined herein, eachcarrier must also be “acceptable” in the sense of being compatible withthe other ingredients of the formulation. The pharmaceutical formulationcontains a compound of the invention in combination with one or morepharmaceutically acceptable ingredients. The carrier can be in the formof a solid, semi-solid or liquid diluent, cream or a capsule. Thesepharmaceutical preparations are a further object of the invention.Usually the amount of active compounds is between 0.1-95% by weight ofthe preparation, preferably between 0.2-20% by weight in preparationsfor parenteral use and preferably between 1 and 50% by weight inpreparations for oral administration. For the clinical use of themethods of the present invention, targeted delivery compositions can beformulated into pharmaceutical compositions or pharmaceuticalformulations for parenteral administration, e.g., intravenous; mucosal,e.g., intranasal; enteral, e.g., oral; topical, e.g., transdermal;ocular, e.g., via corneal scarification or other mode of administration.The pharmaceutical composition comprises an LF polypeptide and at leastone target antigen that is not covalently bound to the LF polypeptide,and in some embodiments, in combination with one or morepharmaceutically acceptable ingredients. The carrier can be in the formof a solid, semi-solid or liquid diluent, cream or a capsule.

As used herein, the term “an intact cell” refers to a living cell withan unbroken, uncompromised plasma membrane, which cell has adifferential membrane potential across the membrane, with the inside ofthe cell being negative with respect to the outside of the cell.

As used herein, the term “N-glycosylated” or “N-glycosylation” refers tothe covalent attachment of a sugar moiety to asparagine residues in apolypeptide. Sugar moieties can include but are not limited to glucose,mannose, and N-acetylglucosamine. Modifications of the glycans are alsoincluded, e.g., siaylation. The LFn polypeptide has threeN-glycosylation sites: asparagine positions 62, 212, and 286 in the 809amino acid polypeptide.

As used herein, the terms “N-glycosylated LFn-fusion polypeptide,”“N-glycosylated LF-fusion polypeptide” or “N-glycosylated fusedpolypeptide” refer to a fusion polypeptide, as defined herein, that hasat least one sugar moiety covalently attached to an asparagine residue.For example, Asn-62, Asn-212, and Asn-286 can be glycosylated in anN-glycosylated LF-fusion polypeptide.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment. Other than inthe operating examples, or where otherwise indicated, all numbersexpressing quantities of ingredients or reaction conditions used hereinshould be understood as modified in all instances by the term “about.”The term “about” when used in connection with percentages can mean ±1%.The present invention is further explained in detail by the followingexamples, but the scope of the invention should not be limited thereto.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims.

Methods and Compositions Involving Lfn Adjuvant

The various components required to perform the methods described hereinand considerations for various aspects of the methods and compositionsare described in the following sections.

I. LF Polypeptides

By way of background and without wishing to be limited by theory, B.anthracis is the causative agent of anthrax in animals and humans. Thetoxin produced by B. anthracis consists of two bipartite proteinexotoxins, lethal toxin (LT) and edema toxin. LT is composed ofprotective antigen (PA) and lethal factor (LF), whereas edema toxinconsists of PA and edema factor (EF). None of these three components,PA, LF, and EF, alone is toxic. Once combined however, edema toxincauses edema and LT causes death by systemic shock in animals andhumans. Consistent with its critical role in forming both toxins, PA hasbeen identified as the protective component in vaccines against anthrax.The molecular mechanism of anthrax toxin action is as follows: PA is a735-amino acid polypeptide that binds to the surface of mammalian cellsby cellular receptors. Once bound, PA is activated by proteolyticcleavage by cellular proteases to a 63-kDa molecule capable of forming aring-shaped heptamer in the plasma membrane of the targeted cell(FIG. 1) (Milne et al., (1994) J. Biol. Chem. 269, 20607-20612, Petosa,et al., (1997) Nature (London) 385, 833-838). The PA heptamer then bindseither EF or LF, which are internalized by endocytosis. After endosomalacidification, PA enables EF or LF to enter the cytosol, presumably bymeans of a pore formed by the heptamer. Within the cytosol, EF acts asan adenylate cyclase (Leppla, S. H. (1982) Proc. Natl. Acad. Sci. USA79, 3162-3166) to convert ATP to cAMP. Abnormally elevated levels ofcAMP perturb cellular metabolism.

Anthrax lethal factor or LF is a protein encoded by GenBank Accessionnumber M29081 (GeneID No: 14343) that is naturally produced by B.anthracis and that has MAPKK protease activity. Deletion analysis of LFshows that the PA binding domain is located within the amino-terminus ofLFn, and that mutational studies demonstrate the PA binding domain islocated within the region of 34 to 254 of the LF polypeptide of SEQ IDNO: 1, and within the region of 34 to 288 of the LF polypeptide of SEQID NO: 2 (Arora et al., J. Biol. Chem. 268:3334 3341 (1993); Milne, etal., (1995) Mol. Microbiol. 15, 661-66).

The action of LF in the cytosol causes the death of host cells by amechanism that is not well understood. LF induces over-production of anumber of lymphokines (Klimpel, et al., (1994) Mol. Microbiol. 13,1093-1100), contributing to lethal systemic shock in host animals.Recent studies also show that LF has two enzymatic activities: it canact as a zinc metalloprotease (Duesbery, et al., (1998) Science 280,734-737), and it inactivates mitogen-activated protein kinase (Hanna, etal.,. (1994) Mol. Med. 1, 7-18). Although it is still not clear howthese two enzymatic activities of LF are connected, both are requiredfor LF toxicity. It has previously been reported that anthrax toxin Bmoieties can be used to deliver eptiopes which in turn elicit anantibody response by the immune system, in the presence of PA (WO97/23236).

B. anthracis LF is a 796-aa polypeptide, and the functional domain forboth enzymatic activities is located between amino acids 383 and 796 ofSEQ ID NO: 1. The N-terminal truncated LF without this catalytic domaincompletely lacks any toxic effect when mixed with PA and added tocultured macrophages or when injected into animals. It does, however,still bind to PA effectively. The PA binding domain of LFn occurs withinresidues 34-288 of SEQ ID NO: 2 (Milne, et al., (1995) Mol. Microbiol.15, 661-66).

The 83 kDa PA polypeptide binds at its carboxyl-terminus to a cellsurface receptor, where it is specifically cleaved by a protease, e.g.,furin, clostripain, or trypsin. This enzymatic cleavage releases a 20kDa amino-terminal PA fragment, while a 63 kDa carboxyl-terminal PAfragment remains bound to the cell surface receptor. The 63 kDa fragmentis also referred to as “processed protective antigen.” Processed PAcontains both a cell surface receptor binding site at itscarboxyl-terminus and a lethal factor binding site at its newamino-terminus (see, e.g., Singh et al., J. Biol. Chem. 264:19103 19107(1989)). Processed PA can be produced by enzymatic cleavage in vitro, exvivo or in vivo, or as a recombinant protein. As used herein the term PArefers PA molecules that have the lethal factor binding site, e.g.,recombinant PA, naturally occurring PA, functional equivalents of PAthat contain the lethal factor binding site, and PA fusion proteins thatcontain the lethal factor binding site.

II. Compositions Comprising LF Polypeptides and Target Antigen

The inventors have established that a fragment of the lethal factor (LF)polypeptide of Bacillus anthracis (B. anthracis) can deliver a fusedtarget antigen to the cytosol of an intact cell. In particular, theinventors have previously demonstrated that in the absence of PA, atarget antigen which is covalently attached (i.e. by a covalent bond orfused) to an LF polypeptide such as LFn or a fragment thereof can beused to deliver an antigen to the cytosol of an intact, living cell andelicit a CTL response to the fused antigen. Surprisingly, the inventorsherein have discovered that it is not necessary for the target antigento be fused to an LF polypeptide to be delivered to the cell cytosol inthe absence of PA. Thus, the inventors have now surprisingly discoveredthat LF polypeptides, such as LFn and fragments or variants thereof canbe used to deliver non-linked (i.e. non-fused) target antigens to thecytosol of a cell in the absence of PA. Accordingly, one aspect of thepresent invention described herein relates to the use of LFpolypeptides, such as LFn or fragments or variants of LFn as an immuneadjuvant to deliver non-linked (i.e. non-fused) antigens to the cytosolof a cell to elicit a CMI response against the antigen.

The methods, compositions and kits described herein employ an LFpolypeptide to deliver a target antigen to the cytosol of a cell from asubject. The LF polypeptide compositions involved generally comprise anLF polypeptide and a target antigen, where the LF polypeptide, e.g., LFnis not covalently linked to target antigen.

Alternatively, in some embodiments of this and other aspects describedherein, the LF polypeptide can be in a non-covalently linked complex orbe associated with the target antigen in some way, for example, to forman LFn:target antigen complex, where the LFn and target antigen areassociated by forces other than a covalent bond, such as van der Waalsforces, electrostatic forces and the like. In some embodiments of thisand other aspects described herein, the composition comprises an LFpolypeptide:target antigen complex, where the LF polypeptide, e.g., LFn,is directly associated with the target antigen by van der Waals forcesor other non-covalent interactions. In alternative embodiments, thecomposition comprises an LF polypeptide:target antigen complex, wherethe LF polypeptide, e.g., an LFn polypeptide is indirectly associatedwith the target antigen, for example by interaction of the LFnpolypeptide with at least a third entity or moiety, and the targetantigen also interacts with a separate portion of the third entity (thatinteracts with the LF polypeptide).

In some embodiments of this and other aspects described herein, thecomposition comprises an LF polypeptide or LFn polypeptide and a targetantigen, where the LF polypeptide is not covalently linked to the targetantigen but the LF polypeptide is non-covalently associated or complexedwith the target antigen in some way. For example, to form an LFn:targetantigen complex. In some embodiments, the composition comprises anLFn:target antigen complex, where the LFn (or fragment or variantthereof) is directly associated with the target antigen by van der Waalsforces or other non-covalent interactions. In alternative embodiments,the composition comprises an LFn:target antigen complex, where the LFn(or fragment or variant thereof) is indirectly associated with thetarget antigen, such as for example by interaction of the LFn (orfragment or variant thereof) with at least one third moiety, and thetarget antigen interacts with the same third moiety that interacts withthe LFn polypeptide. Such interactions can be any non-covalent bondassociation known by a skilled artisan, such as, for example but notlimited to, van der Waals forces, hydrophilic interactions, hydrophobicinteractions and other non-covalent interactions. In some embodiments,at least one, or at least two, or at least 3, or at least 4 or morethird entities can be used to associate LFn (or a fragment or variantthereof) with the target antigen. For example, the present inventioncomprises compositions which comprise complexes such as, anLFn:moiety:target antigen complex, or Lfn:moiety:moiety:target antigencomplex, Lfn:moiety:moiety:moiety:target antigen complex, and such likecomplexes. In some embodiments, a moiety which associates with LFn canbe the same or different from a moiety which binds with the targetantigen, and all the moieties can be the same within a complex, ordifferent within the complex.

Alternatively, in this aspect and all other aspects described herein,the present invention also encompasses a complex where a moiety iscovalently linked to either (but not both simultaneously) an LFpolypeptide or a target. For example, a target antigen can be covalentlybonded (e.g. fused) to a moiety, and the moiety can interact vianon-covalent interactions with the LF polypeptide such that the targetantigen and LF polypeptide form a complex. Conversely, an LF polypeptidecan be covalently bonded (e.g. fused) to a moiety, and the moiety caninteract via non-covalent interactions with the target antigen such thatthe target antigen and LF polypeptide form a complex. Importantly, whilean LF polypeptide and target antigen can not be covalently linked to thesame moiety, they can be covalently linked to different moieties whichnon-covalently interact with each other, i.e., an LF polypeptide can becovalently linked to moiety A, and a target antigen can be covalentlylinked to moiety B, and moiety-A can interact with moiety B vianon-covalent interactions, to form a LF-moiety-A:moiety-B-target antigencomplex.

A. Lethal Factor (LF) of Bacillus anthracis and the N-terminal Fragment(LFn)

As discussed briefly above, anthrax Lethal Factor or LF is a protein,encoded by GenBank Accession Number M29081 (Gene ID No: 143143), that isnaturally produced by B. anthracis and that has MAPKK protease activity.Deletion analysis of LF shows that the PA binding domain is locatedwithin the amino-terminus of LFn. Mutational studies demonstrate the PAbinding domain is located within the region of 34 to 254 of the LFpolypeptide of SEQ ID NO: 1, and within the region of 34 to 288 of theLF polypeptide of SEQ ID NO: 2 (Arora et al., J. Biol. Chem. 268:33343341 (1993); Milne, et al., (1995) Mol. Microbiol. 15, 661-66). Thethree-dimensional atomic resolution structures of LF have now beensolved by X-ray crystallography. Andrew D. Pannifer et. al., describesthe crystal structure of LF and its complex with a 16-amino acid residue(16-mer) peptide representing the N-terminus of its natural substrate,MAPKK-2, in Nature vol. 414, pg. 229-233 (2001) as a protein thatcomprises four structural domains: domain I binds themembrane-translocating component of anthrax toxin, the protectiveantigen (PA); domains II, III and IV together create a long deep groovethat holds the 16-residue N-terminal tail of MAPKK-2 before cleavage.Domain I is perched on top of the other three domains, which areintimately connected and comprise a single folding unit. The onlycontacts between domain I and the rest of the molecule are with domainII, and these chiefly involve charged polar and water-mediatedinteractions. The nature of the interface is consistent with the abilityof a recombinant N-terminal fragment (residues 1-254, excluding thesignal peptide) to be expressed as a soluble folded domain thatmaintains the ability to bind PA and enables the translocation ofheterologous fusion proteins into the cytosol (Ballard, J. D., et. al.,1996, Proc. Natl Acad. Sci. USA 93, 12531-12534; Goletz, T. J. et al.,1997, Proc. Natl Acad. Sci. USA 94, 12059-12064). Moreover, deletion ofthe first 36 residues of LFn had no effect on its binding to PA or LFability to be translocated across membranes (D. Borden Lacy, et.al.,2002, J. Biol. Chem., 277:3006-3010). Domain I consists of a 12-helixbundle that packs against one face of a mixed four-stranded β-sheet,with a large (30-residue) ordered loop, L1, between the second and third-strands forming a flap over the distal face of the sheet (see FIG. 1).The exact docking site on domain I for PA is unknown, but the integrityof the folded domain seems to be required, because a series of insertionand point mutants of buried residues in domain I that presumably disruptthe fold abrogate binding of PA and toxicity (Quinn, C. P., et. al.,1991, J. Biol. Chem., 266: 20124-20130; Gupta, P., et. al., 2001,Biochem. Biophys. Res. Comm , 280:158-163). In addition, LFn has beenshown to deliver exogenous protein antigens to the majorhistocompatibility complex class I pathway in the cytosol of B-cells,CTL-cells and macrophages in the absence of PA (Huyen Cao, et. al.,2002, The Journal of Infectious Diseases; 185:244-251; N. Kushner, et.al., 2003, Proc Natl Acad Sci U S A. 100: 6652-6657). The PA-independentLFn mediated delivery of target antigen polypeptide depends onfunctional transport-associated proteins for intracellular antigenprocessing and transport into the endoplasmic reticulum for binding toMHC class I molecules.

An abrupt turn at the end of the last helix of domain I leads directlyinto the first helix of domain II (residues 263-297 and 385-550).Although sequence-based comparisons failed to yield any homology, thestructural similarity with the catalytic domain of the B. cereus toxin,VIP2 (Protein Data Bank accession code 1QS2), is outstanding. Domain IIand VIP2 superimpose with an RMSD of 3.3 Å and a sequence identity of15%, as determined by DALI (Holm, L. & Sander, 1997, Nucleic Acids Res.25, 231-234). VIP2 contains an NAD-binding pocket and conserved residuesinvolved in NAD binding and catalysis. Domain II lacks these conservedresidues; moreover, a critical glutamic acid that is conservedthroughout the family of ADP ribosylating toxins (Carroll, S. F. &Collier, R. J., 1984, Proc. Natl Acad. Sci. USA 81, 3307-3311) isreplaced by a lysine (K518). We therefore expect that domain II does nothave ADP-ribosylating activity.

Domain III is a small α-helical bundle with a hydrophobic core (residues303-382), inserted at a turn between the second and third helices ofdomain II. Sequence analysis has revealed the presence of a 101-residuesegment comprising five tandem repeats (residues 282-382), and suggestedthat repeats 2-5 arose from a duplication of repeat 1. The crystalstructure reveals that repeat 1 actually forms the second helix-turnelement of domain II, whereas repeats 2-5 form the four helix-turnelements of the helical bundle, suggesting a mechanism of creating a newprotein domain by the repeated replication of a short segment of theparent domain. Domain III is required for LF activity, because insertionmutagenesis and point mutations of buried residues in this domainabrogate function (Quinn, C. P., et. al., 1991, J. Biol. Chem. 266,20124-20130). It makes limited contact with domain II, but shares ahydrophobic surface with domain IV. Its location is such that itseverely restricts access to the active site by potential substratessuch as the loops of a globular protein; that is, it contributes towardsspecificity for a flexible ‘tail’ of a protein substrate. It alsocontributes sequence specificity by making specific interactions withthe substrate (see below).

Domain IV (residues 552-776) consists of a nine-helix bundle packedagainst a four-stranded -sheet. Sequence comparisons had failed todetect any homology with other proteins of known structure beyond theHExxH motif. The three-dimensional structure reveals that the β-sheetand the first six helices can be superimposed with those of themetalloprotease thermolysin, with an RMSD of 4.9 Å over 131 residues.Large insertions and deletions occur elsewhere within the loopsconnecting these elements, so that the overall shapes of the domains arequite different. In particular, a large ordered loop (L2) insertedbetween strands 42 and 43 of the sheet partly obscures the active site,packs against domain II, and provides a buttress for domain III.

A zinc ion (Zn2+) is coordinated tetrahedrally by a water molecule andthree protein side chains, in an arrangement typical of the thermolysinfamily. Two coordinating residues are the histidines from the HExxHmotif (His 686 and His 690) lying on one helix (44), as expected. Thestructure reveals that the third coordinating residue is Glu 735 fromhelix 46. Glu 687 from the HExxH motif lies 3.5 Å from the watermolecule, well positioned to act as a general base to activate thezinc-bound water during catalysis. The hydroxyl group of a tyrosineresidue (Tyr 728) forms a strong hydrogen bond (O—O distance 2.6 Å) tothe water molecule, on the opposite side of Glu 687, and probablyfunctions as a general acid to protonate the amine leaving group.

B. anthracis encodes an 809 amino acid LF polypeptide. The mature B.anthracis LF is a 796 amino acid polypeptide produced by cleavage of theN-terminal leader peptide. The functional domain for both enzymaticactivities is located between amino acids 383 and 796 of SEQ ID NO: 1.The N-terminal truncated LF without this catalytic domain completelylacks any toxic effect when mixed with PA and added to culturedmacrophages or when injected into animals. It does, however, still bindto PA effectively. The PA binding domain of LFn occurs within residues34-288 of SEQ ID NO:2 (Milne, et al., (1995) Mol. Microbiol. 15,661-66).

The gene encoded 809 amino acid polypeptide B. anthracis LF has sevenpotential N-glycosylation sites located at asparagine positions 62, 212,286, 478, 712 736, and 757. Within the LFn (1-288), there are threepotential N-glycosylation sites, at asparagine positions 62, 212, and286, all of which have the potential of >0.51 according to the NetNGlyc1.0 Prediction software from the Technical University of Denmark. TheNetNglyc server predicts N-Glycosylation sites in proteins usingartificial neural networks that examine the sequence context ofAsn-Xaa-Ser/Thr sequons.

The term “LFn polypeptide” includes LF polypeptide fragments representedby SEQ ID NOs 3 and 4, as well as recombinant LFn, and functional LFnequivalents, fragments, and variants that retain the function to deliverthe polypeptide target antigen (that is not covalently linked to the LFnpolypeptide) to the cytosol of an intact cell, preferably a living cell.The term “LFn polypeptide” therefore includes functional LFn homologuessuch as polymorphic variants, alleles, mutants, and closely relatedinterspecies variants that have at least about 60% amino acid sequenceidentity to LFn and have the function to deliver polypeptide targetantigen that is not covalently linked to the LFn polypeptide to thecytosol of a cell, as determined using the assays described herein. Inparticular embodiments, the LFn polypeptides are substantially identicalto LFn of SEQ ID NO: 3 and SEQ ID NO: 4 as disclosed herein. In someembodiments, some functional polymorphic variants, alleles, mutants, andclosely related interspecies variants of LFn that function to deliver apolypeptide target antigen to an intact cell can be determined by themethods and assays as disclosed in U.S. patent application Ser. No.10/473,190 which is incorporated herein by reference.

In some embodiments, an LFn mimetic is useful in the compositions andmethods described herein. An “LFn mimetic” refers to a compound ormolecule, e.g., a peptide, polypeptide, or small chemical molecule thatfunctions as LFn to deliver a target antigen to the cytosol of a cell toinduce a CMI response against the antigen. LFn mimetics thus include LFnhomologues. LFn mimetics would also include small LFn peptides thatretain the LFn function to deliver polypeptide antigens (not linked tothe LFn mimetic) to the cytosol of the cell, and conservativelysubstituted variants thereof, as well as truncated versions of LFn thatretain ability of LFn to deliver polypeptide antigens (not linked to theLFn mimetic) to the cytosol of a cell. LFn mimetics are tested usingassays for a CMI response to the target antigen as disclosed herein andin the Examples of U.S. patent application Ser. No. 10/473,190 (which isincorporated herein in its entirety by reference), e.g., induction of aCTL response to the delivered target antigen. When testing for an LFnmimetic, LFn is typically used as a positive control for delivery of thetarget antigen to a cell.

While the whole of the N-terminal amino acid residues 1-288 (i. e.domain I, see FIG. 3) of the LF polypeptide promotes the transmembranedelivery of other proteins, it should be understood that smallerfragments of domain I can be sufficient to translocate across cellmembrane and promote the transmembrane delivery of other proteins whennon-covalently linked to the LF polypeptide. The x-ray crystal structureof domain I shows 12 alpha helices and four beta sheet secondary proteinstructure. Smaller fragments of domain I of an LF polypeptide thatpreserve alpha helices and/or beta sheet secondary protein structures ofdomain I can be used to translocate across cell membrane and promote thetransmembrane delivery of other non-covalently linked proteins (i.e.target antigens). One skilled in the art can determine the presence ofalpha helices and beta sheet secondary protein structure in an LFpolypeptide using methods known in the art, such as circular dichroism(CD).

One aspect described herein is a means for eliciting a specific immuneresponse, in particular a cell mediated cytotoxic immune response (CMI)to a target antigen, whereby a target antigen is delivered to thecytosol of a cell by being present in a non-covalently-linked formcomposition comprising an LF polypeptide, such as an LFn polypeptide ora fragment or variant thereof. In some embodiments of this and otheraspects described herein, a preferred protein for delivery of non-fused(i.e. non-covalently linked) target antigens to the cytosol of a cell isan N-terminal fragment of the lethal Factor, herein referred to “LFn”and corresponds to amino acid SEQ ID NO: 4.

In some embodiments, the present invention relates to a means to elicitan immune response to a target antigen, where the target antigen is notfused to LFn, and where LFn contacts the target antigen and transducesthe target antigen to the cytosol of a cell in the absence of PA.

One aspect of the present invention relates to a composition comprisingan LFn polypeptide, or a homologue or fragment thereof and a targetantigen, where the LFn is not covalently linked to the target antigen,and the LFn polypeptide or fragment thereof is not directly linked tothe target antigen. In some embodiments, the composition does notcomprise PA.

The inventors have discovered that a fragment of LFn which is at leastabout 250 amino acids or less, or at least about 150 amino acids orless, or at least about 104 amino acids or less, is able to deliver atarget antigen to the cell and is useful in the methods and compositionsdescribed herein.

In one embodiment, the LFn polypeptide used in the delivery of thetarget antigen polypeptide comprises at least the 60 carboxy-terminalamino acids of SEQ. ID. No. 3, or a conservative substitution variantthereof. In another embodiment, the LFn polypeptide consists essentiallyof 60 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservativesubstitution variant thereof. In yet another embodiment, the LFnpolypeptide consists of 60 carboxy-terminal amino acids of SEQ. ID. No.3, or a conservative substitution variant thereof.

In one embodiment, the LFn polypeptide used to deliver the targetantigen polypeptide comprises at least the 80 carboxy-terminal aminoacids of SEQ. ID. No. 3, or a conservative substitution variant thereof.In another embodiment, the LFn polypeptide consists essentially of 80carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservativesubstitution variant thereof. In yet another embodiment, the LFnpolypeptide consists of 80 carboxy-terminal amino acids of SEQ. ID. No.3, or a conservative substitution variant thereof.

In one embodiment, the LFn polypeptide used to deliver the targetantigen polypeptide comprises at least the 104 carboxy-terminal aminoacids of SEQ. ID. No. 3, or a conservative substitution variant thereof.In another embodiment, the LFn polypeptide consists essentially of 104carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservativesubstitution variant thereof. In yet another embodiment, the LFnpolypeptide consists of 104 carboxy-terminal amino acids of SEQ. ID. No.3, or a conservative substitution variant thereof.

In one embodiment, the LFn polypeptide used to deliver the targetantigen polypeptide consists of the amino acid sequence corresponding toSEQ. ID. No. 5, or a conservative substitution variant thereof. Inanother embodiment, the LFn polypeptide consists essentially of theamino acid sequence corresponding to SEQ. ID. No. 5, or a conservativesubstitution variant thereof. In yet another embodiment, the LFnpolypeptide comprises of the amino acid sequence corresponding to SEQ.ID. No. 5, or a conservative substitution variant thereof.

In one embodiment, the LFn polypeptide used to deliver the targetantigen polypeptide comprises the amino acid sequence corresponding toSEQ. ID. No. 4, or a conservative substitution variant thereof. Inanother embodiment, the LFn polypeptide consists essentially of theamino acid sequence corresponding to SEQ. ID. No. 4, or a conservativesubstitution variant thereof. In yet another embodiment, the LFnpolypeptide consists of the amino acid sequence corresponding to SEQ.ID. No. 4, or a conservative substitution variant thereof.

In one embodiment, the LFn polypeptide used to deliver the targetantigen polypeptide comprises the amino acid sequence corresponding toSEQ. ID. No. 3, or a conservative substitution variant thereof. Inanother embodiment, the LFn polypeptide consists essentially of theamino acid sequence corresponding to SEQ. ID. No. 3, or a conservativesubstitution variant thereof. In yet another embodiment, the LFnpolypeptide consists of the amino acid sequence corresponding to SEQ.ID. No. 3, or a conservative substitution variant thereof.

In some embodiments, an LFn polypeptide as described herein comprises anon-functional binding site for PA, and thus is a mutant of LFn whichdoes not result in functional binding with PA. Such mutants include, butare not limited to mutants altered at one or more of the residuescritical for interacting with PA, such as a mutation in one or more ofthe following residues: Y22; L188; D187; Y226; L235; H229 (see Lacy etal., J. Biol. Chem., 2002; 277; 3006-3010); D106A; Y108K; E135K; D136K;N140A and K143A (see Melnyk et al., J. Biol. Chem., 2006; 281; 1630-1635and Cunningham et al., PNAS, 2002; 99; 70497052, which are incorporatedherein in their entirety by reference).

LFn polypeptides as described herein, or a conservative substitutionvariants thereof, promote transmembrane delivery of a target antigenthat is not covalently linked to the LFn polypeptide. Methods ofdetermining membrane translocation are well known in the art, asdescribed, for example, in Wesche, J., et. al., 1998, Biochemistry 37:15737-15746 and Sellman, B. R.,et. al., 2001 J. Biol. Chem. 276:8371-8376. By way of brief explanation, CHO-K1 cells in a 24-well plateare chilled on ice, washed, and incubated on ice for 2 h with any of theLF polypeptide (or a conservative substitution variant thereof orfragments of domain I) and a target antigen as described herein thathave been labeled with [35S]methionine in an in vitrotranscription/translation system (Promega). The cells then are washedwith ice-cold PBS at pH 5.0 or 8.0, incubated at 37° C. for 1 min, andeither treated with Pronase to digest residual untranslocated 35S at thecell surface or left untreated as controls. The cells are then lysed,and 35S liberated into the lysis buffer is assayed. The percenttranslocation is defined as dpm protected from Pronase/dpm bound tocells x 100. The cell lysate of cells incubated with LF polypeptides orfragments of domain I that facilitate transmembrane delivery would havehigher percent translocation.

Alternatively, a target antigen and/or an LFn polypeptide can bemodified or labeled to allow each to be monitored for transmembranedelivery. For example, an LF polypeptide (such as LFn, LF or smallerfragments of domain I) can be fused to a fluorescent molecule, such as agreen fluorescent protein which is useful to assay for membranetranslocation capability, as described in N. Kushner, et. al., 2003,Proc Natl Acad Sci U S A. 100: 6652-6657. Briefly, HeLa cells (AmericanType Culture Collection) are grown on collagen-treated chamber slides(BD Science) to reach ˜80% confluence and incubated with 40 μg/m1purified GFP or LFn-GFP at 37° C. for 1 or 2 h. After washing, GFPfluorescence is compared between GFP and GFP-LFn treated samples.Membrane translocation is evidenced by GFP signal greater in theLFn-GFP-treated cells than in cells treated with GFP alone. Someincubations can also be performed in the presence of 100 μg/m1 Texasred-conjugated transferrin (Invitrogen Inc., Molecular Probes) as amarker for the endocytic pathway. For the transferrin experiments, cellsare washed four times with cold DMEM and then fixed for 15 min in 4%paraformaldehyde in cold PBS. For antibody labeling, slides are thenincubated on ice for 15 min in 50 mM NH₄Cl in PBS and then in PBScontaining 0.1% saponin for 20 min on ice. After further washing in PBS,slides are incubated at room temperature for 1 hr in a moisture chamberwith PBS containing 4% donkey serum and the following primaryantibodies: mouse anti-early endosome antigen 1 (EEA-1) (BD Laboratory)to stain early endosomes, mouse anti-Lamp1 and anti-Lamp2 (DevelopmentalStudies Hybridoma Banks, University of Iowa, Iowa City) to stain lateendosomes and lysosome, mouse Ab-1 (Oncogene) to stain the Golgiapparatus, mouse anti-mitochondrial antibody from Calbiochem, and rabbitanti-calreticulin (StressGen Biotechnologies, Victoria, Canada). Cellsare then processed for secondary antibody staining and microscopy.Fusion LFn-GFP that promotes transmembrane delivery would be visualizedin the interior of the cell. The antibody markers will further indicatesub-cellular localization of the translocated GFP.

As discussed above briefly, in one embodiment, an LFn polypeptide usefulin the compostions and methods described herein does not bind B.anthracis protective antigen (PA) protein. The PA protein is the naturalbinding partner of LF, forming bipartite protein exotoxin, lethal toxin(LT). The PA protein is a 735-amino acid polypeptide, a multi-functionalprotein that binds to cell surface receptors, mediates the assembly andinternalization of the complexes, and delivers them to the host cellendosome. Once PA is attached to the host receptor, it is cleaved by ahost cell surface (furin family) protease before it is able to bind LF.The cleavage of the N-terminus of PA enables the C-terminal fragment toself-associate into a ring-shaped heptameric complex (prepore) that canbind LF and delivers LF into the cytosol. The N-terminal fragment(residues 1-288, domain I) can be expressed as a soluble folded domainthat maintains the ability to bind PA and enables the translocation ofnon-covalently linked target antigen proteins into the cytosol. Smallerfragments of this residue 1-288 N-terminal fragment have been shown toalso translocate heterologous fusion proteins into the cytosol in theabsence of PA. Hence, in one embodiment, smaller fragments of LFdescribed herein can translocate across membranes but do not bind PA.Methods of measuring or detecting protein-protein interaction are wellknown. One skilled in the art can determine PA binding activity, forexample, by mixing and incubating PA63 with LFn for a period of time,chemically cross-linking of any complex formed and analysis of thecovalently linked complex by gel electrophoresis or by radioactivitycounting as described by Quinn C P. et. al., 1991, J. Biol. Chem.266:20124-20130. Briefly, the binding assay is determined at 5° C. bycompetition with radiolabeled 125 I-LFn. Native LF or full-lengthN-terminal (amino acid 1-288) LFn is radiolabeled (˜7.3×106 cpm/μgprotein) using Bolton-Hunter reagent (Amersham Corp). For bindingstudies, J774A.1 cells cultured in 24-well tissue culture plates arecooled by incubating at 4° C. for 60 min and then placing the plates onice. The medium is then replaced with cold (4° C.) minimal essentialmedium containing Hanks' salts (GIBCO/BRL) supplemented with 1% (w/v)bovine serum albumin and 25 mM HEPES (binding medium). Native PA (0.1g/ml) is added with radiolabeled native LF (125I-LF, 0.1 μg/ml, 7.3×10⁶cpm/μg) and the plates incubated for 14 h on wet ice. Mutant LF proteinsare assayed at varying concentrations for their ability to compete withnative 125I-LF. For quantitation of bound, radiolabeled LF, cells aregently washed twice in cold binding medium, once in cold Hanks' balancedsalt solution, solubilized in 0.50 ml of 0.1 M NaOH, and counted in agamma counter (Beckman Gamma 9000).

In one embodiment, an LFn polypeptide substantially lacks the aminoacids 1-33 of SEQ. ID. No. 3. Amino acids 1-33 of SEQ. ID. No. 3encompass the signal peptide that is predicted to direct thepost-translational transport of the LF protein. In some embodiments, anLFn polypeptide lacks a signal peptide that functions to direct thepost-translational transport of the LF polypeptide. In otherembodiments, an LFn polypeptide comprises a signal peptide forco-translation on the ER. The signal peptide is also called a leaderpeptide in the N- terminus, which can or can not be cleaved off afterthe translocation through the ER membrane. One example of a signalpeptide is MAPFEPLASGILLLLWLIAPSRA (SEQ ID NO: 6). Other examples ofsignal peptides can be found at SPdb, a Signal Peptide Database, whichis found at the world wide web site of http colon “forward slash”“forward slash” proline “dot” bic “dot” nus “dot” edu “dot” sg “forwardslash” spdb “forward slash”.

In some embodiments, an LFn polypeptide as described herein is immunesilent, or substantially inert, meaning that the LFn polypeptide doesnot function as an immunogen (i.e. it is not a target antigen) and doesnot substantially generate a CMI response to itself.

In one embodiment, the LF polypeptide is N-linked glycosylated.N-glycosylation is important for the folding of some eukaryoticproteins, providing a co-translational and post-translationalmodification mechanism that modulates the structure and function ofmembrane and secreted proteins. Glycosylation is the enzymatic processthat links saccharides to produce glycans, and attaches them to proteinsand lipids. In N-glycosylation, glycans are attached to the amidenitrogen of asparagine side chain during protein translation. The threemajor saccharides forming glycans are glucose, mannose, andN-acetylglucosamine molecules. The N-glycosylation consensus isAsn-Xaa-Ser/Thr, where Xaa can be any of the known amino acids. Oneskilled in the art can use bioinformatics software such as NetNGlyc 1.0Prediction software from the Technical University of Denmark to find theN-glycosylation sites in an LF polypeptide of the present invention. TheNetNglyc server predicts N-Glycosylation sites in proteins usingartificial neural networks that examine the sequence context ofAsn-Xaa-Ser/Thr sequons. The NetNGlyc 1.0 Prediction software can beaccessed at the EXPASY website. In one embodiment, N-glycosylationoccurs in the target antigen polypeptide as described herein. In anotherembodiment, N-glycosylation occurs in an LF polypeptide such as an LFnpolypeptide as described herein, for example, at asparagine positions62, 212, and/or 286, all of which have the potential of >0.51 accordingto the NetNGlyc 1.0 Prediction software. Various combinations ofN-glycosylation in LF polypeptides of the present invention arepossible. In some embodiments, an LF polypeptide described herein has asingle N-glycosylation at one of these three sites: asparagine positions62, 212, and 286. In some other embodiments, an LF polypeptide describedherein is N-glycosylated at two of these three sites: asparaginepositions 62, 212, and 286. In another embodiment, an LF polypeptidedescribed herein is N-glycosylated at all three sites: asparaginepositions 62, 212, and 286. In yet another embodiment, N-glycosylationoccurs in both the target antigen polypeptide and the LFn polypeptide.In some embodiments, the glycans of the LF polypeptide and/or targetantigen as described herein are modified, for example, sialyated orasialyated. Glycosylation analysis of proteins is known in the art. Forexample, via glycan hydrolysis (using enzymes such as N-glycosidase F,EndoS endoglycosidase, sialidase or with 4N trifluroacetic acid),derivitization, and chromatographic separation such as LC-MS or LC-MS/MS(Pei Chen et. al., 2008, J. Cancer Res. Clin. Oncology, 134: 851-860;Kainz, E. et. al., 2008, Appl Environ Microbiol., 74: 1076-1086).

The gene encoded 809-aa polypeptide B. anthracis LF is not predicted tohave any O-glycosylation sites according to the NetOGlyc 3.1 Predictionsoftware from the Technical University of Denmark. The NetOglyc serverproduces neural network predictions of mucin type GalNAc O-glycosylationsites in proteins. However, in some embodiments, the LFn used herein isN-glycosylated.

In one embodiment, the LFn and/or antigen compositions described hereincomprise glycosylated proteins. In other words, the LF, LFn or thetarget antigens can each be glycosylated proteins, e.g., with O-linkedglycosylation or N-linked glycosylation. In yet another embodiment, theLF, LFn or the target antigens can be both O-linked and N-linkedglycosylated. In other embodiments, other types of glycosylations arepossible, e.g. C-mannosylation. In one embodiment of the vaccinecompositions described herein, the LFn polypeptide is N-glycosylated.Glycosylation of proteins occurs predominantly in eukaryotic cells.N-glycosylation is important for the folding of some eukaryoticproteins, providing a co-translational and post-translationalmodification mechanism that modulates the structure and function ofmembrane and secreted proteins. Glycosylation is the enzymatic processthat links saccharides to produce glycans, and attaches them to proteinsand lipids. In N-glycosylation, glycans are attached to the amidenitrogen of asparagine side chain during protein translation. The threemajor saccharides forming glycans are glucose, mannose, andN-acetylglucosamine molecules. The N-glycosylation consensus isAsn-Xaa-Ser/Thr, where Xaa can be any of the known amino acids. O-linkedglycosylation occurs at a later stage during protein processing,probably in the Golgi apparatus. In O-linked glycosylation,N-acetyl-galactosamine, O-fucose, O-glucose, and/or N-acetylglucosamineis added to serine or threonine residues. One skilled in the art can usebioinformatics software such as NetNGlyc 1.0 and NetOGlyc Predictionsoftwares from the Technical University of Denmark to find the N- andO-glycosylation sites in a polypeptide in the present invention. TheNetNglyc server predicts N-Glycosylation sites in proteins usingartificial neural networks that examine the sequence context ofAsn-Xaa-Ser/Thr sequons. The NetNGlyc 1.0 and NetOGlyc 3.1 Predictionsoftware can be accessed at the EXPASY website. In one embodiment,N-glycosylation occurs in the target antigen polypeptide of the fusionpolypeptide described herein. In another embodiment, N-glycosylationoccurs in the LFn polypeptide of a fusion polypeptide described herein,for example, at asparagine positions 62, 212, and/or 286, all of whichhave the potential of >0.51 according to the NetNGlyc 1.0 Predictionsoftware.

Various combinations of N-glycosylation in the fusion polypeptide of thepresent invention are possible. In some embodiments, the individual andfusion polypeptides described herein have a single N-glycosylation atone of these three sites: asparagine positions 62, 212, and 286 of LFn.In other embodiments, the individual and fusion polypeptides describedherein are N-glycosylated at two of these three sites: asparaginepositions 62, 212, and 286 of LFn. In another embodiment, the individualand fusion polypeptides described herein is N-glycosylated at all threesites: asparagine positions 62, 212, and 286 of LFn. In yet anotherembodiment, N-glycosylation occurs in both the target antigenpolypeptide and the LFn polypeptide. In some embodiments, the glycans ofthe LFN and target antigent polypeptides described herein are modified,for example, sialyated or asialyated. Glycosylation analysis of proteinsis known in the art, for example, via glycan hydrolysis (using enzymessuch as N-glycosidase F, EndoS endoglycosidase, sialidase or with 4Ntrifluroacetic acid), derivitization, and chromatographic separationsuch as LC-MS or LC-MS/MS (Pei Chen et. al., 2008, J. Cancer Res.Clin.Oncology, 134: 851-860; Kainz,E. et. al., 2008, Appl. Environ.Microbiol., 74: 1076-1086). LFn is predicted to have no O-linkedglycosylation sites of >0.50 potential.

In one embodiment, an LFn polypeptide as described herein is expressedbacterial cells and purified from a protein expression system using hostcells selected from the group consisting of: mammalian cells, insectcells, yeast cells, and plant cells. The cloning, protein expression,and purification of recombinant proteins are known. One skilled in theart can use modern molecular techniques to construct an isolatedpolynucleotide encoding any of the LF polypeptides described herein, andligate the isolated polynucleotide into a vector to form a recombinantvector, wherein the recombinant vector is an expression vector that iscompatible with a protein expression system using host cells selectedfrom the group consisting of: bacterial cells; mammalian cells; insectcells; yeast cells; and plant cells. Thus, mammalian cells, insectcells, yeast cells and plant cells are preferred. It is preferred thatthe host cell can N-glycosylate the recombinant LF polypeptide. Thereare many options for an expression vector depending on the choice ofprotein expression system and the types of host cells used. In oneembodiment, the recombinant vector is a viral vector, such as, arecombinant baculovirus vector, an adeno-associated virus (AAV) vectoror a lentivirus vector. Viral vectors provide ease of introducing thecoding polynucleotide construct into the desired host cells. Forexample, adeno-associated virus (AAV) vector or a lentivirus vectorinfects mammalian cells and baculovirus vectors infect lepidopteraninsect cells, such as, Spodoptera frugiperda cells. Expression of an LFnpolypeptide as described herein in eukaryotic host cells, e. g,mammalian cells and insect cells can result in N-glycosylation of theLFn polypeptide thus expressed.

B. Production of LF Polypeptides

i. Expression Systems:

Recombinant proteins, such an LF polypeptide as described herein can bereadily produced by routine methods by one or ordinary skill in the art,such as by routine techniques in the field of recombinant genetics.Basic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994)).

Nucleic acid sequences encoding an LF polypeptide which is notcommercially available can be chemically synthesized according to thesolid phase phosphoramidite triester method first described by Beaucage& Caruthers, Tetrahedron Letts. 22:1859 1862 (1981), using an automatedsynthesizer, as described in Van Devanter et. al., Nucleic Acids Res.12:6159 6168 (1984). Purification of oligonucleotides is by eithernative acrylamide gel electrophoresis or by anion-exchange HPLC asdescribed in Pearson & Reanier, J. Chrom. 255:137 149 (1983). Thesequence a synthetic oligonucleotide or a cloned gene encoding an LFpolypeptide can be verified after cloning using, e.g., the chaintermination method for sequencing double-stranded templates of Wallaceet al. Gene 16:21 26 (1981).

A. Cloning Methods for the Isolation of Nucleotide Sequences EncodingLFn.

In general, the nucleic acid sequences encoding an LF polypeptide suchas LFn can be cloned from cDNA and genomic DNA libraries or isolatedusing amplification techniques with oligonucleotide primers. Forexample, LFn sequences are typically isolated from B. anthracis nucleicacid (genomic or cDNA) libraries.

The coding DNA sequences are typically cloned into intermediate vectorsbefore transformation into prokaryotic or eukaryotic cells forreplication and/or expression. These intermediate vectors are typicallyprokaryote vectors, e.g., plasmids, or shuttle vectors, as describedbelow.

Amplification techniques using primers can also be used to amplify andisolate LFn coding sequences from DNA or RNA (see U.S. Pat. Nos.4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)). Methods such as polymerasechain reaction (PCR) and ligase chain reaction (LCR) can be used toamplify nucleic acid sequences of LF directly from mRNA, from cDNA, fromgenomic libraries or cDNA libraries, and from plasmids. Degenerateoligonucleotides can be used to amplify homologues. These primers can beused, e.g., to amplify a probe of several hundred nucleotides, which isthen used to screen a human library for full-length LF, which can bethen be used to generate LFn. Alternatively, the nucleic acid for LFncan be directly amplified.

Nucleic acids encoding an LF polypeptide such as LFn can also beisolated from expression libraries using antibodies as probes. Syntheticoligonucleotides can be used to construct recombinant LFn genes for useas probes, for expression of protein, and for construction ofpolymorphic variants or mutants such as deletion mutants. This method isperformed using a series of overlapping oligonucleotide: usually 40 120by in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.

Polymorphic variants, alleles, and interspecies homologues that aresubstantially identical to LFn can be isolated using LFn nucleic acidprobes and oligonucleotides under stringent hybridization conditions, byscreening libraries using probes, or using amplification techniques asdescribed above. Alternatively, expression libraries can be used toclone polymorphic variant, alleles, and interspecies homologues, bydetecting expressed homologues immunologically with antisera or purifiedantibodies, which also recognize and selectively bind to the homologue.

The gene encoding LF has been cloned and sequenced, and has beenassigned Genebank accession no. M29081 (Robertson & Leppla, Gene 44:7178 (1986); Bragg & Robertson, Gene 81:45 54 (1989); see also U.S. Pat.Nos. 5,591,631, 5,677,274; see generally Leppla, Anthrax Toxins, inBacterial Toxins and Virulence Factors in Disease (Moss et al., eds.,1995)).

The nucleic acids of interest are typically cloned into intermediatevectors before transformation into prokaryotic or eukaryotic cells forreplication and/or expression. These intermediate vectors are typicallyprokaryote vectors, e.g., plasmids, or shuttle vectors, as describedbelow.

To obtain high level expression of a cloned gene, such as those cDNAsencoding LFn, one typically subclones the nucleic acid into anexpression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the LFn protein areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229 235 (1983); Mosbach et al., Nature 302:543 545 (1983)). Kitsfor such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application and is not critical. Exemplarypromoters include the SV40 early promoter, SV40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells, as well as prokaryoticpromoters. The promoter is preferably positioned about the same distancefrom the heterologous transcription start site as it is from thetranscription start site in its natural setting. As is known in the art,however, some variation in this distance can be accommodated withoutloss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the nucleic acid inhost cells. A typical expression cassette thus also contains signalsrequired for efficient polyadenylation of the transcript, ribosomebinding sites, and translation termination. The nucleic acid sequenceencoding the gene of choice can typically be led to a cleavable signalpeptide sequence to promote secretion of the encoded protein by thetransformed cell. Such signal peptides would include, among others, thesignal peptides from tissue plasminogen activator, insulin, and neurongrowth factor, and juvenile hormone esterase of Heliothis virescens.Additional elements of the cassette can include enhancers and, ifgenomic DNA is used as the structural gene, introns with functionalsplice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region can beobtained from the same gene as the promoter sequence or can be obtainedfrom different genes.

Additional elements that are typically included in expression vectorsalso include a replicon that functions in E. coli, a gene encodingantibiotic resistance to permit selection of bacteria that harborrecombinant plasmids, unique restriction sites in nonessential regionsof the plasmid to allow insertion of eukaryotic sequences. In addition,some expression systems have markets that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. The particular antibiotic resistance genechosen is not critical, any of the many resistance genes known in theart are suitable. The prokaryotic sequences are preferably chosen suchthat they do not interfere with the replication of the DNA in eukaryoticcells, if necessary.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells can be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and, in someinstances fusion expression systems such as GST and LacZ. Otherexemplary eukaryotic vectors include pMSG, pAV009/A.sup.+,pMTO10/A.sup.+, pMAMneo-5, baculovirus pDSVE. Tags can also be added torecombinant proteins to provide convenient methods of isolation, e.g.,c-myc, or hexahistidine.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of LFn protein,which are then purified using standard techniques (see.e.g. Colley etal. J. Biol. Chem. 264:17619 17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques, e.g., calcium phosphate transfection,polybrene, protoplast fusion, electroporation, liposomes,microinjection, plasma vectors, viral vectors (see, e.g., Morrison, J.Bact. 132:349 351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347 362 (Wu et al., eds, 1983).

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe gene of choice, which is recovered from the culture using standardtechniques identified below.

LF polypeptides as disclosed herein e.g., an LFn polypeptide can all besynthesized and purified by protein and molecular methods that are wellknown to one skilled in the art. Preferably molecular biology methodsand recombinant heterologous protein expression systems are used. Forexample, recombinant protein can be expressed in mammalian, insect,yeast, or plant cells.

Some examples of recombinant cloning and truncation of LF, LFn, theirexpression, and specific site mutations and insertions are described,for example, in WO/2002/079417, WO/2008/048289, US patent No.20040166120, Huyen Cao, et. al., 2002, The Journal of InfectiousDiseases;185:244-251; N. Kushner, et. al., 2003, Proc Natl Acad Sci U SA. 100: 6652-6657; Ballard, J. D., et. al., 1996, Proc. Natl Acad. Sci.USA 93, 12531-12534; and Goletz, T. J. et al., 1997, Proc. Natl Acad.Sci. USA 94, 12059-12064, which are incorporated herein by reference intheir entirety. Approaches similar to those described in thesereferences can be used to produce an LF polypeptide, such as LFnpolypeptide as described herein.

In one embodiment, an isolated polynucleotide encoding an LFpolypeptide, such as LFn polypeptide described herein, can be producedby conventional polymerase chain reaction (PCR) cloning techniques ascommonly known by persons of ordinary skill in the art. In someembodiments, an LF polypeptide and a target antigen can be expressedfrom the same expression vector, where each polynucleotide (i.e. oneencoding the LF polypeptide and one encoding the target antigen) areseparated so they are expressed as separate (non-covalently linked)proteins. Typically, the polynucleotide encoding an LF polypeptide andthe polynucleotide encoding a target antigen are separated by an IRES(internal Ribosome Entry site) nucleic acid sequence. A polynucleotideencoding an LF polypeptide (and/or a target antigen polynucleotide) canbe cloned into a general purpose cloning vector such as pUC19, pBR322,pBluescript vectors (Stratagene Inc.) or pCR TOPO® from Invitrogen Inc.The resultant recombinant vector carrying the nucleic acid encoding anLF polypeptide, such as LFn polypeptide as described herein can then beused for further molecular biological manipulations such assite-directed mutagenesis to create a variant LF polypeptide asdescribed herein or can be subcloned into protein expression vectors orviral vectors for protein synthesis in a variety of protein expressionsystems using host cells selected from the group consisting of mammaliancell lines, insect cell lines, yeast, bacteria, and plant cells.

Each PCR primer should have at least 15 nucleotides overlapping with itscorresponding templates at the region to be amplified. The polymeraseused in the PCR amplification should have high fidelity such asStrategene's PfuUltra™ polymerase for reducing sequence mistakes duringthe PCR amplification process. For ease of ligating several separate PCRfragments together, for example in the construction of an LFpolypeptide, such as LFn polypeptide, and subsequently inserting into acloning vector, the PCR primers should also have distinct and uniquerestriction digestion sites on their flanking ends that do not anneal tothe DNA template during PCR amplification. The choice of the restrictiondigestion sites for each pair of specific primers should be such thatthe LF polypeptide, such as LFn polypeptide, coding DNA sequence isin-frame and will encode the LF polypeptide from beginning to end withno stop codons. At the same time the chosen restriction digestion sitesshould not be found within the coding DNA sequence for the LFnpolypeptide. The coding DNA sequence for the fusion polypeptide can beligated into cloning vector pBR322 or one of its derivatives, foramplification, verification of fidelity and authenticity of the chimericcoding sequence, substitutions/or specific site-directed mutagenesis forspecific amino acid mutations and substitutions in the LF polypeptide.

Alternatively the coding DNA sequence for an LF polypeptide, such as anLFn polypeptide, can be PCR cloned into a vector using for example,Invitrogen's TOPO® cloning method in topoisomerase-assisted TA vectorssuch as pCR®-TOPO, pCR®-Blunt II-TOPO, pENTR/D-TOPO®, andpENTR/SD/D-TOPO®. Both pENTR/D-TOPO®, and pENTR/SD/D-TOPO® aredirectional TOPO entry vectors which allow the cloning of the DNAsequence in the 5′→3′ orientation into a Gateway® expression vector.Directional cloning in the 5′→3′ orientation facilitates theunidirectional insertion of the DNA sequence into a protein expressionvector such that the promoter is upstream of the 5′ ATG start codon ofthe LF polypeptide coding DNA sequence, enabling promoter driven proteinexpression. The recombinant vector carrying the coding DNA sequence forthe LF polypeptide can be transfected into and propagated in generalcloning E. coli such as XL1Blue, SURE (Stratagene) and TOP-10 cells(Invitrogen).

Standard techniques known to those of skill in the art can be used tointroduce mutations (to create amino acid substitutions in thepolypeptide sequence of an LF polypeptide, e.g., an LFn polypeptidedescribed, i. e. SEQ. ID. No. 3 or 4 or 5) in the nucleotide sequenceencoding an LFn polypeptide, e.g., SEQ. ID. No. 3 and 4, including, forexample, site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, the variant LF polypeptide has less than 50 amino acidsubstitutions, less than 40 amino acid substitutions, less than 30 aminoacid substitutions, less than 25 amino acid substitutions, less than 20amino acid substitutions, less than 15 amino acid substitutions, lessthan 10 amino acid substitutions, less than 5 amino acid substitutions,less than 4 amino acid substitutions, less than 3 amino acidsubstitutions, or less than 2 amino acid substitutions relative to a LFor LFn polypeptide.

Certain silent or neutral missense mutations can also be made in the DNAcoding sequence that do not change the encoded amino acid sequence orthe capability to promote transmembrane delivery. These types ofmutations are useful to optimize codon usage, or to improve recombinantprotein expression and production.

Specific site-directed mutagenesis of a coding sequence for apolypeptide in a vector can be used to create specific amino acidmutations and substitutions. Site-directed mutagenesis can be carriedout using, e. g. the QUIKCHANGE® site-directed mutagenesis kit fromStratagene according to the manufacturer's instructions.

In one embodiment, disclosed herein are expression vectors comprisingthe coding DNA sequence for an LF polypeptide, e.g., an LFn polypeptidedescribed herein for the expression and purification of the recombinantLF polypeptide produced from a protein expression system using hostcells selected from, e.g., mammalian, insect, yeast, or plant cells. Theexpression vector should have the necessary 5′ upstream and 3′downstream regulatory elements such as promoter sequences, ribosomerecognition and TATA box, and 3′ UTR AAUAAA (SEQ ID NO: 11)transcription termination sequence for efficient gene transcription andtranslation in its respective host cell. The expression vector can haveadditional sequence such as 6X-histidine, V5, thioredoxin,glutathione-S-transferase, c-Myc, VSV-G, HSV, FLAG, maltose bindingpeptide, metal-binding peptide, HA and “secretion” signals (e.g.,Honeybee melittin, PHO, Bip), which are incorporated into the expressedLF polypeptide. In addition, there can be enzyme digestion sitesincorporated after these sequences to facilitate enzymatic removal ofthem after they are not needed. These additional sequences are usefulfor the detection of the LF polypeptide expression, for proteinpurification by affinity chromatography, enhanced solubility of therecombinant protein in the host cytoplasm, and/or for secreting theexpressed LF polypeptide out into the culture media, or the spheroplastof the yeast cells. The expression of an LF polypeptide can beconstitutive in the host cells or it can be induced, e.g., with coppersulfate, sugars such as galactose, methanol, methylamine, thiamine,tetracycline, infection with baculovirus, and(isopropyl-beta-D-thiogalactopyranoside) IPTG, a stable synthetic analogof lactose.

In one embodiment, a recombinant vector comprising an LF polypeptidesuch as an LFn polypeptide described herein is an expression vector thatfacilitates protein expression. In another embodiment, the expressionvector comprising an LF polypeptide described herein is a viral vector,such as adenovirus, adeno-associated virus (AAV), retrovirus,baculovirus and lentivirus vectors, among others. The expression vectorscan be viral or non-viral vectors. Recombinant viruses provide aversatile system for gene expression studies and therapeuticapplications.

The LF polypeptide, e.g., an LFn polypeptide described herein can beexpressed in a variety of expression host cells e.g., yeasts, mammaliancells, insect cells and plant cells such as Chlamadomonas, or even incell-free expression systems. From the cloning vector, the nucleic acidcan be subcloned into a recombinant expression vector that isappropriate for the expression of an LF polypeptide in mammalian,insect, yeast, or plant cells or a cell-free expression system such as arabbit reticulocyte expression system.

Subcloning can be achieved by PCR cloning, restriction digestionfollowed by ligation, or recombination reaction such as those of thelambda phage-based site-specific recombination using the GATEWAY® LR andBP CLONASE™ enzyme mixtures. Subcloning should be unidirectional suchthat the 5′ ATG start codon of the nucleic acid is downstream of thepromoter in the expression vector. Some vectors are designed to transfercoding nucleic acid for expression in mammalian cells, insect cells andyear in one single recombination reaction. For example, some of theGATEWAY® (Invitrogen) destination vectors are designed for theconstruction of baculovirus, adenovirus, adeno-associated virus (AAV),retrovirus, and lentiviruses, which upon infecting their respective hostcells, permit heterologous expression of an LF polypeptide in theappropriate host cells. Transferring a gene into a destination vector isaccomplished in just two steps according to manufacturer's instructions.There are GATEWAY® expression vectors for protein expression in insectcells, mammalian cells, and yeast. Following transformation andselection in E. coli, the expression vector is ready to be used forexpression in the appropriate host.

Examples of other expression vectors and host cells are the strong CMVpromoter-based pcDNA3.1 (Invitrogen) and pClneo vectors (Promega) forexpression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat,and MCF-7; replication incompetent adenoviral vector vectors pAdeno X,pAd5F35, pLP-Adeno-X-CMV (Clontech), pAd/CMV/V5-DEST, pAd-DEST vector(Invitrogen) for adenovirus-mediated gene transfer and expression inmammalian cells; pLNCX2, pLXSN, and pLAPSN retrovirus vectors for usewith the Retro-X™ system from Clontech for retroviral-mediated genetransfer and expression in mammalian cells; pLenti4/V5-DEST™,pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ (Invitrogen) forlentivirus-mediated gene transfer and expression in mammalian cells;adenovirus-associated virus expression vectors such as pAAV-MCS,pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) for adeno-associatedvirus-mediated gene transfer and expression in mammalian cells; BACpak6baculovirus (Clontech) and pFastBac™ HT (Invitrogen) for the expressionin Spodopera frugiperda 9 (Sf9), Sf11, Tn-368 and BTI-TN-5B4-1 insectcell lines; pMT/BiP/V5-His (Invitrogen) for the expression in DrosophilaSchneider S2 cells; Pichia expression vectors pPICZα, pPICZ, pFLDα andpFLD (Invitrogen) for expression in Pichia pastoris and vectors pMETαand pMET for expression in P. methanolica; pYES2/GS and pYD1(Invitrogen) vectors for expression in yeast Saccharomyces cerevisiae.Recent advances in the large scale expression heterologous proteins inChlamydomonas reinhardtii are described by Griesbeck C. et. al. 2006Mol. Biotechnol. 34:213-33 and Fuhrmann M. 2004, Methods Mol Med.94:191-5. Foreign heterologous coding sequences are inserted into thegenome of the nucleus, chloroplast and mitochodria by homologousrecombination. The chloroplast expression vector p64 carrying the mostversatile chloroplast selectable marker aminoglycoside adenyltransferase (aadA), which confer resistance to spectinomycin orstreptomycin, can be used to express foreign protein in the chloroplast.The biolistic gene gun method can be used to introduce the vector in thealgae. Upon its entry into chloroplasts, the foreign DNA is releasedfrom the gene gun particles and integrates into the chloroplast genomethrough homologous recombination.

In some instances, a suitable system for expressing an LF polypeptide,such as an LFn polypeptide as described herein includes a baculovirusexpression system (i.e. a BEVS system). In such a bacliovirus expressionsystem, the first step is the construction of a recombinant baculovirusvector, either by homologous recombination or by site specifictransposition. To obtain a recombinant baculovirus vector by homologousrecombination, a baculovirus transfer vector is needed. A baculovirustransfer vector is a temporary vector whose sole purpose is to enablethe insertion of foreign coding DNA, under an appropriate gene promoter,into the baculovirus genome at a site that would not affect normal viralreplication. The baculovirus transfer vector comprises a portion of thebaculovirus genomic sequence that spans the intended insertion site ofthe foreign coding DNA. The most common regions contain the polyhedrinor p10 gene. Both are dispensable for viral replication in cell cultureand insect larvae and the production of infectious extracellular virus.Both proteins are highly expressed at a very late phase of viralreplication and effect high level of transcription of the foreign genewhen inserted back into the viral genome. A typical baculovirus transfervector comprises a promoter, a transcriptional terminator, and mostoften native viral sequences and regions flanking both sides of thepromoter that are homologous to the target genes in the viral genome.The region between the promoter and the transcriptional terminator canhave multiple restriction enzyme digestion sites for facilitatingcloning of the foreign coding sequence, in this instance, the coding DNAsequence for an LF polypeptide, e.g., an LFn polypeptide and a targetantigen. Additional sequences can be included, e.g., signal peptidesand/or tag coding sequences (such as His-tag, MAT Tag, FLAG tag)recognition sequence for enterokinase, honeybee melittin secretionsignal, beta-galactosidase, glutathione S-transferase (GST) tag upstreamof the MCS) for facilitating the secretion, identification, properinsertion, positive selection of recombinant virus, and/or purificationof the recombinant protein. Subsequent to the construction of thebaculovirus transfer vector, it is mixed with AcNPV viral DNA andco-transfected into insect cells to establish an infection. The nativepolyhedrin gene is removed by a double-cross over homologousrecombination event and replaced by the foreign coding sequence to beexpressed in the insect cells. Inactivation of the polyhedrin gene bydeletion or by insertion results in mutants that do not produceocclusions in infected cells. These occlusion-negative viruses formplaques that are different from plaques produced by wild-type viruses,and this distinctive plaque morphology is useful as a means to screenfor recombinant viruses.

In one embodiment, an LF polypeptide described herein can be produced byexpression from a recombinant baculoviruses vector. In anotherembodiment, any LF polypeptide described herein is expressed by aninsect cell. In yet another embodiment, any LF polypeptide describedherein is isolated from an insect cell. There are several benefits ofprotein expression with baculovirus in insect cells, including highexpression levels, ease of scale-up, production of proteins withposttranslational modifications, and simplified cell growth. Insectcells do not require CO₂ for growth and can be readily adapted tohigh-density suspension culture for large-scale expression. Many of thepost-translational modification pathways present in mammalian systemsare also utilized in insect cells, allowing the production ofrecombinant protein that is antigenically, immunogenically, andfunctionally similar to the native mammalian protein.

As background only, and without wishing to be bound by theory,baculoviruses are DNA viruses in the family Baculoviridae. These virusesare known to have a narrow host-range that is limited primarily toLepidopteran species of insects (butterflies and moths). The baculovirusAutographa californica Nuclear Polyhedrosis Virus (AcNPV), which hasbecome the prototype baculovirus, replicates efficiently in susceptiblecultured insect cells. AcNPV has a double-stranded closed circular DNAgenome of about 130,000 base-pairs and is well characterized with regardto host range, molecular biology, and genetics. Many baculoviruses,including AcNPV, form large protein crystalline occlusions within thenucleus of infected cells. A single polypeptide, referred to as apolyhedrin, accounts for approximately 95% of the protein mass of theseocclusion bodies. The gene for polyhedrin is present as a single copy inthe AcNPV viral genome. Because the polyhedrin gene is not essential forvirus replication in cultured cells, it can be readily modified toexpress foreign genes. The foreign gene sequence is inserted into theAcNPV gene just 3′ to the polyhedrin promoter sequence such that it isunder the transcriptional control of the polyhedrin promoter. TheBaculovirus Expression Vector System (BEVS) is a safe and rapid methodfor the abundant production of recombinant proteins in insect cells andinsects pioneered in the laboratory of Dr. Max D. Summers.

Baculovirus expression systems are powerful and versatile systems forhigh-level, recombinant protein expression in insect cells. Expressionlevels up to 500 mg/1 have been reported using the baculovirusexpression system, making it an ideal system for high-level expression.Recombinant baculoviruses that express foreign genes are constructed byway of homologous recombination between baculovirus DNA and chimericplasmids containing the gene sequence of interest. Recombinant virusescan be detected by virtue of their distinct plaque morphology andplaque-purified to homogeneity.

Baculoviruses are particularly well-suited for use as eukaryotic cloningand expression vectors. They are generally safe by virtue of theirnarrow host range which is restricted to arthropods. The U.S.Environmental Protection Agency (EPA) has approved the use of threebaculovirus species for the control of insect pests. AcNPV has beenapplied to crops for many years under EPA Experimental Use Permits.AcNPV wild type and recombinant viruses replicate in a variety of insectcells, including continuous cell lines derived from the fall armyworm,Spodoptera frugiperda (Lepidoptera; Noctuidae). S. frugiperda cells havea population doubling time of 18 to 24 hours and can be propagated inmonolayer or in free suspension cultures.

In one embodiment of this and other aspects as described herein, theinvention provides a composition for raising or detecting acell-mediated immune (CMI) response to a target antigen polypeptide, thecomposition comprising an LF polypeptide such as an LFn polypeptide or afragment thereof which is not linked to a target antigen, oralternatively, is non-covalently linked to the target antigen andwhereby the LF polypeptide promotes the transmembrane delivery of thetarget antigen to the cytosol of an intact cell.

A good number of baculovirus transfer vectors and the correspondingappropriately modified host cells are commercially available, forexample, pAcGP67, pAcSECG2TA, pVL1392, pVL1393, pAcGHLT, and pAcAB4 fromBD Biosciences; pBAC-3, pBAC-6, pBACgus-6, and pBACsurf-1 from Novagen,and pPolh-FLAG and pPolh-MAT from Sigma Aldrich. One skilled in the artwould be able to clone and ligate the coding region of the Bacillusanthracis lethal factor N-terminal (LFn) portion or fragment thereofusing specially designed oligonucleotide probes and polymerase chainreaction (PCR) methodologies that are well known in the art. One skilledin the art would also be able to clone and ligate the coding sequencefor an LF polypeptide into a selected baculovirus transfer vector. Thecoding sequences of LFn and the target antigen polypeptide or fragmentthereof should be ligated in-frame and the chimeric coding sequenceshould be ligated downstream of the promoter, and between the promoterand the transcription terminator. Subsequent to that, the recombinantbaculovirus transfer vector is transfected into regular cloningEscherichia coli, such as XL1Blue. Recombinant transfer vector DNA isthen selected by antibiotic resistance to remove any non-recombinantplasmid DNA and subsequently amplified and purified for transfectioninto Spodoptera frugiperda (SF) cells.

As an example, the oligonucleotide5′-GGAGGAACATATGGCGGGCGGTCATGGTGATG-3′ (SEQ. ID. NO.9) is used tointroduce an Ndel site and serve as a forward primer in theamplification of the coding DNA sequence for LFn-(amino acids 1-263) andthe oligonucleotide 5′-CTAGGATCCTTACCGTTGATCTTTAAGTTCTTCC-3′ (SEQ. ID.NO.10) is used to introduce a BamHI site and act as the reverse primer.PCR amplification is performed using the cDNA template according toGenBank Accession No. M29081. The forward primers for LFn-(28-263),LFn-(33-263), LFn-(37-263), LFn-(40-263), and LFn-(43-263) can bedesigned accordingly permit the PCR amplification of the coding sequenceof the appropriate truncated LFn and also introduce an NdeI site.Accordingly, the polynucleotide coding sequence can be easily producedwith a NdeI restriction site at the 5′ and a BamHI restriction site athe 3′ end, allowing unidirectional cloning into an appropriateexpression cloning vector, such as a baculovirus expression vector. Thesequences can introduce a stop sequence (TAA) after the coding region ofthe LF sequence. The common BamHI site at the end of the amplifiedcoding sequence of LF facilitates the ligation of the amplified codingsequences into an appropriate expression vector, such as a selectedbaculovirus transfer vector that has Ndel and BamHI sites with theappropriate orientation. The newly constructed baculovirus transfervector can be transformed into Escherichia coli DHS. E. colitransformants can be screened by digestion and verified by sequencing.After that, the baculovirus transfer vector can be isolated forco-transfection into insect cells for homologous recombination.

To obtain a recombinant baculovirus vector by site specifictransposition, e. g. with Tn7 to insert foreign genes into bacmid DNApropagated in E. coli., Invitrogen Inc. provides the pFASTBACTM plasmidand bacmid containing DH10BAC™ competent E. coli for constructing arecombinant baculovirus vector by site specific transposition. Thecoding sequence is cloned into a pFASTBAC™ plasmid and the recombinantplasmid is transformed into an DH10BAC™ competent E. coli harboringbacmid, baculovirus shuttle vector, with a mini-attTn7 target site and ahelper plasmid. The mini-attTn7 element on the pFASTBAC™ plasmid cantranspose to the mini-attTn7 target site on the bacmid in the presenceof transposition proteins provided by the helper plasmid. Coloniescontaining recombinant bacmids are identified by antibiotics selectionand by blue/white screening, since the transposition results in thedisruption of the LacZ gene that is flanked by the mini-attTn7 targetsite on the bacmid. The bacmid is then harvested for transfection ofinsect cells.

In some instances, specific site-directed mutagenesis of the chimericcoding sequence in the baculovirus transfer vector can be performed tocreate specific amino acid mutations and substitutions to furtherpromote transmembrane delivery, protein expression or protein folding.Examples of amino acid substitutions include glutamate for aspartate.Site-directed mutagenesis can be carried out, e.g., using theQUIKCHANGE® site-directed mutagenesis kit from Stratagene according tomanufacture's instructions or any methods known in the art.

Standard viral DNA is used to co-transfect S. frugiperda (SF) cells.Putative recombinant viruses containing the recombinant molecules areisolated from the virus expressed from these transfected monolayers.Because the polyhedrin structural gene has been removed, plaquescontaining the recombinant viruses can be easily identified since theylack occlusion bodies. Confirmation that these recombinants contain thedesired chimeric coding sequence is established by methods well known inthe art, such as hybridization with specific gene probes, plaque assays,and end point dilution.

A preferred host cell line for protein production from recombinantbaculoviruses described herein is Sf900+. Another preferred host cellline for protein production from recombinant baculoviruses is Sf9.Sf900+ and Sf9 are non-transformed, non-tumorigenic continuous celllines derived from the fall armyworm, Spodoptera frugiperda(Lepidoptera; Noctuidae).

Sf900+ and Sf9 cells are propagated at 28±2° C. without carbon dioxidesupplementation. The culture medium used for Sf9 cells is TNMFH, asimple mixture of salts, vitamins, sugars and amino acids, supplementedwith 10% fetal bovine serum. Aside from fetal bovine serum, no otheranimal derived products (i.e, trypsin, etc.) are used in cellpropagation. Serum free culture medium (available as Sf900 culturemedia, Gibco BRL, Gaithersburg, Md.) can also be used to grow Sf9 cellsand is preferred for propagation of Sf900+ cells. Sf9 cells have apopulation doubling time of 18-24 hours and can be propagated inmonolayer or in free suspension cultures. S. frugiperda cells have notbeen reported to support the replication of any known mammalian viruses.

Plaque assays of baculovirus transfected monolayers SF cells are wellknown in the art. Once recombinant baculoviral vectors that express theproteins are established, then the virus can be amplified and purifiedfor infection of SF cells.

Purification of Virus. Viral particles produced from the first passageare purified from the media using a known purification method such assucrose density gradient centrifugation. For example, virus is harvested24-48 hours post infection by centrifuging media of infected cells. Theresulting viral pellet is resuspended in buffer and centrifuged througha buffered sucrose gradient. The virus band is harvested from the 40-45%sucrose region of the gradient, diluted with buffer and pelleted bycentrifugation at 100,000×g. The purified virus pellet is resuspended inbuffer and stored at −70° C. or used in large scale infection of cellsfor protein production.

The infection process, including viral protein synthesis, viral assemblyand partial cell lysis can be complete by approximately 72 hourspost-infection. This can be protein dependent and thus can occur earlieror later. The proteins produced in infected cells can radiolabeled with³⁵S-methionine, ³H-leucine, or ³H-mannose and both cell-associated andcell-free polypeptides can be analyzed by electrophoresis onpolyacrylamide gels to determine their molecular weight. The expressionof these products can also be examined at different timespost-infection, prior to cell lysis.

In some embodiments, an LF polypeptide as described herein can beexpressed from viral infection of mammalian cells. The viral vectors canbe, for example, adenovirus, adeno-associated virus (AAV), retrovirus,and lentivirus. A simplified system for generating recombinantadenoviruses is presented by He TC. et. al. Proc. Natl. Acad. Sci. USA95:2509-2514, 1998. The gene of interest is first cloned into a shuttlevector, e.g. pAdTrack-CMV. The resultant plasmid is linearized bydigesting with restriction endonuclease Pme I, and subsequentlycotransformed into E. coli. BJ5183 cells with an adenoviral backboneplasmid, e.g. pAdEasy-1 of Stratagene's AdEasy™ Adenoviral VectorSystem. Recombinant adenovirus vectors are selected for kanamycinresistance, and recombination confirmed by restriction endonucleaseanalyses. Finally, the linearized recombinant plasmid is transfectedinto adenovirus packaging cell lines, for example HEK 293 cells(E1-transformed human embryonic kidney cells) or 911 (E1-transformedhuman embryonic retinal cells) (Human Gene Therapy 7:215-222, 1996).Recombinant adenovirus are generated within the HEK 293 cells.

Recombinant lentivirus has the advantage of delivery and expression ofan LF polypeptide, e.g., an LFn polypeptide that is not linked, or notcovalently linked to a target antigen in either dividing andnon-dividing mammalian cells. The HIV-1 based lentivirus can effectivelytransduce a broader host range than the Moloney Leukemia Virus(MoMLV)-base retroviral systems. Preparation of the recombinantlentivirus can be achieved using, for example, the pLenti4/V5-DEST™,pLenti6/V5-DEST™ or pLenti vectors together with ViraPower™ LentiviralExpression systems from Invitrogen.

Recombinant adeno-associated virus (rAAV) vectors are applicable to awide range of host cells including many different human and non-humancell lines or tissues. rAAVs are capable of transducing a broad range ofcell types and transduction is not dependent on active host celldivision. High titers, >10⁸ viral particle/ml, are easily obtained inthe supernatant and 10¹¹-10¹² viral particle/ml with furtherconcentration. The transgene is integrated into the host genome soexpression is long term and stable.

Large scale preparation of AAV vectors is made by a three-plasmidcotransfection of a packaging cell line: AAV vector carrying the codingnucleic acid, AAV RC vector containing AAV rep and cap genes, andadenovirus helper plasmid pDF6, into 50×150 mm plates of subconfluent293 cells. Cells are harvested three days after transfection, andviruses are released by three freeze-thaw cycles or by sonication.

AAV vectors can be purified by two different methods depending on theserotype of the vector. AAV2 vector is purified by the single-stepgravity-flow column purification method based on its affinity forheparin (Auricchio, A., et. al., 2001, Human Gene therapy 12; 71-6;Summerford, C. and R. Samulski, 1998, J. Virol. 72:1438-45; Summerford,C. and R. Samulski, 1999, Nat. Med. 5: 587-88). AAV2/1 and AAV2/5vectors are currently purified by three sequential CsC1 gradients.

ii. Protein Purification Systems:

An LF polypeptide, e.g., an LFn polypeptide described herein can beexpressed and purified by a variety methods known to one skilled in theart. Recombinant LF or LFn polypeptides, for example, can be purifiedfrom any suitable expression system. Polypeptides can be purified tosubstantial purity by standard techniques, including selectiveprecipitation with such substances as ammonium sulfate; columnchromatography, immunopurification methods, and others (see, e.g.,Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat.No. 4,673,641; Ausubel et al., supra; and Sambrook et al. supra).

The next step is to purify the proteins for uses and compositionsdescribed herein, e. g. evaluation for use as vaccines or screeningagents. If an LF polypeptide described herein is designed with secretionsignal peptides, the encoded polypeptides are often released into thecell culture medium. Media from these infected cells can be concentratedand the proteins purified using standard methods. Salt precipitation,sucrose gradient centrifugation and chromatography, high or fastpressure liquid chromatography (HPLC or FPLC) can be used because thesemethods allow rapid, quantitative and large scale purification ofproteins, and do not denature expressed products.

The efficiency of synthesis of the desired gene product is dependent onmultiple factors including: (1) the choice of an expression vectorsystem; (2) the number of gene copies that will be available in thecells as templates for the production of mRNA; (3) the promoterstrength; (4) the stability and structure of the mRNA; (5) the efficientbinding of ribosomes for the initiation or translation; (6) theproperties of the protein product, such as, the stability of the geneproduct or lethality of the product to the host cells; (7) the abilityof the system to synthesize and export the protein from the cells, thussimplifying subsequent analysis, purification and use.

A number of procedures can be employed when recombinant proteins arepurified. For example, proteins having established molecular adhesionproperties can be reversibly fused to the protein of choice. With theappropriate ligand, the protein can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. Finally,the protein of choice can be purified using affinity or immunoaffinitycolumns.

After the protein is expressed in the host cells, the host cells can belysed to liberate the expressed protein for purification. Methods oflysing the various host cells are featured in “Sample Preparation-Toolsfor Protein Research” EMD Bioscience and in the Current Protocols inProtein Sciences (CPPS). A preferred purification method is affinitychromatography such as metal-ion affinity chromatograph using nickel,cobalt, or zinc affinity resins for histidine-tagged LF polypeptides.Methods of purifying histidine-tagged recombinant proteins are describedby Clontech using their TALON® cobalt resin and by Novagen in their pETsystem manual, 10th edition. Another preferred purification strategy isimmuno-affinity chromatography, for example, anti-myc antibodyconjugated resin can be used to affinity purify myc-tagged LFpolypeptides. When appropriate protease recognition sequences arepresent, LF polypeptides can be cleaved from the histidine or myc tag,releasing the LF polypeptide from the affinity resin while thehistidine-tags and myc-tags are left attached to the affinity resin.Non-tagged LF polypeptides can be affinity purified using copperaffinity chromatography.

Standard protein separation techniques for purifying recombinant andnaturally occurring proteins are well known in the art, e. g. solubilityfractionation, size exclusion gel filtration, and various columnchromatography.

Solubility Fractionation: Often as an initial step, particularly if theprotein mixture is complex, an initial salt fractionation can separatemany of the unwanted host cell proteins (or proteins derived from thecell culture media) from the protein of interest. The preferred salt isammonium sulfate Ammonium sulfate precipitates proteins by effectivelyreducing the amount of water in the protein mixture. Proteins thenprecipitate on the basis of their solubility. The more hydrophobic aprotein is, the more likely it is to precipitate at lower ammoniumsulfate concentrations. A typical protocol includes adding saturatedammonium sulfate to a protein solution so that the resultant ammoniumsulfate concentration is between 20-30%. This concentration willprecipitate the most hydrophobic of proteins. The precipitate is thendiscarded (unless the protein of interest is hydrophobic) and ammoniumsulfate is added to the supernatant to a concentration known toprecipitate the protein of interest. The precipitate is then solubilizedin buffer and the excess salt removed if necessary, either throughdialysis or diafiltration. Other methods that rely on solubility ofproteins, such as cold ethanol precipitation, are well known to those ofskill in the art and can be used to fractionate complex proteinmixtures.

Size Differential Filtration: The molecular weight of the protein ofchoice can be used to isolated it from proteins of greater and lessersize using ultrafiltration through membranes of different pore size (forexample. Amicon or Millipore membranes). As a first step, the proteinmixture is ultrafiltered through a membrane with a pore size that has alower molecular weight cut-off than the molecular weight of the proteinof interest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

Column Chromatography: The protein of choice can also be separated fromother proteins on the basis of its size, net surface charge,hydrophobicity, and affinity for ligands. In addition, antibodies raisedagainst recombinant or naturally occurring proteins can be conjugated tocolumn matrices and the proteins immunopurified. All of these methodsare well known in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).For example, LFn can be purified using a PA63 heptamer affinity column(Singh et al., J. Biol. Chem. 269:29039 29046 (1994)).

In some embodiments, a combination of purification steps comprising, forexample: (i) anion exchange chromatography, (ii) hydroxyapatitechromatography, (iii) hydrophobic interaction chromatography, and (iv)size exclusion chromatography can be used to purify the LF polypeptidesdescribed herein.

Cell-free expression systems are also contemplated. Cell-free expressionsystems offer several advantages over traditional cell-based expressionmethods, including the easy modification of reaction conditions to favorprotein folding, decreased sensitivity to product toxicity andsuitability for high-throughput strategies such as rapid expressionscreening or large amount protein production because of reduced reactionvolumes and process time. The cell-free expression system can useplasmid or linear DNA. Moreover, improvements in translation efficiencyhave resulted in yields that exceed a milligram of protein permilliliter of reaction mix. An example of a cell-free translation systemcapable of producing proteins in high yield is described by Spirin AS.et. al., Science 242:1162 (1988). The method uses a continuous flowdesign of the feeding buffer which contains amino acids, adenosinetriphosphate (ATP), and guanosine triphosphate (GTP) throughout thereaction mixture and a continuous removal of the translated polypeptideproduct. The system uses E. coli lysate to provide the cell-freecontinuous feeding buffer. This continuous flow system is compatiblewith both prokaryotic and eukaryotic expression vectors. Large scalecell-free production of the integral membrane protein EmrE multidrugtransporter is described by Chang G. el. al., Science 310:1950-3 (2005).

Other commercially available cell-free expression systems include theEXPRESSWAY™ Cell-Free Expression Systems (Invitrogen) which utilize anE. coli-based in-vitro system for efficient, coupled transcription andtranslation reactions to produce up to milligram quantities of activerecombinant protein in a tube reaction format; the Rapid TranslationSystem (RTS) (Roche Applied Science) which also uses an E. coli-basedin-vitro system; and the TNT Coupled Reticulocyte Lysate Systems(Promega) which uses rabbit reticulocyte-based in-vitro system.

C. Target Antigens

In one embodiment of this aspect and all other aspects described herein,a target antigen is any antigen whose delivery to the cytosol isdesired. The target antigen is either not linked or non-covalentlylinked to an LF polypeptide. Thus, in some embodiments, antigens whichare associated, for example by some form of non-covalent linkage such aselectrostatic interactions, van der Waals forces etc. are alsoencompassed.

In some embodiments, antigens include viral, bacterial, parasitic, andtumor associated antigens. Preferred viral antigens include proteinsfrom any virus where a cell-mediated immune response is desired.Particularly preferred viruses include HIV-1, HIV-2, hepatitis viruses(including hepatitis B and C), Ebola virus, West Nile virus, and herpesvirus such as HSV-2. Preferred bacterial antigens include those from S.typhi and Mycobacteria (including M. tuberculosis). Preferred parasiticantigens include those from Plasmodium (including P. falciparum). Anantigen can also include, for example, pathogenic peptides, toxins,toxoids, subunits thereof, or combinations thereof (e.g., tetanus,diphtheria toxoid, cholera subunit B, etc.).

In some embodiments, a target antigen polypeptide described herein isany antigen associated with a pathology, for example an infectiousdisease or pathogen, or cancer or an immune disease such as anautoimmune disease. In order to improve the likelihood of producing acell mediated response to the target antigen, the amino acid sequence ofa target antigen polypeptide can be analyzed in order to identifydesired portions of amino acid sequence which can be involved orassociated with receptor binding, such as binding to MHC receptors, orthe target receptor to which the antigen binds. For example, a targetantigen polypeptide sequences can be subjected to computer analysis toidentify such sites.

In some embodiments, a target antigen is a whole virus or an attenuatedvirus, where an attenuated virus is a non-live or inactive virus. Insuch embodiments, a composition comprising an LF polypeptide such as anLFn polypeptide and a target antigen such as a whole virus, such as anattenuated virus which is not linked to the LF polypeptide, the LFpolypeptide functions like a classic adjuvant (i.e. the LF polypeptideenhances the immunological response, such as a CMI response to the wholeand/or attenuated virus target antigen).

One example of an infectious disease antigen is TbH9 (also known as Mtb39A), a tuberculosis antigen. Other tuberculosis antigens include, butare not limited to, DPV (also known as Mtb8.4), 381, Mtb41, Mtb40,Mtb32A, Mtb9.9A, Mtb9.8, Mtb16, Mtb72f, Mtb59f, Mtb88f, Mtb71f, Mtb46fand Mtb31f (“f” indicates that it is a fusion or two or more proteins).

If an antigen is not readily soluble per se, the antigen can be presentin the formulation in a suspension or even as an aggregate. In someembodiments, hydrophobic antigen can be solubilized in a detergent, forexample a polypeptide containing a membrane-spanning domain.Furthermore, for formulations containing liposomes, an antigen in adetergent solution (e.g., a cell membrane extract) can be mixed withlipids, and liposomes then can be formed by removal of the detergent bydilution, dialysis, or column chromatography. Certain antigens such as,for example, those from a virus (e.g., hepatitis A) need not be solubleper se, but can be incorporated directly into a liposome in the form ofa virosome (Morein and Simons, 1985).

Plotkin and Mortimer (1994) provide antigens which can be used tovaccinate animals or humans to induce an immune response specific forparticular pathogens, as well as methods of preparing antigen,determining a suitable dose of antigen, assaying for induction of animmune response, and treating infection by a pathogen (e.g., bacterium,virus, fungus, or parasite).

Target bacteria include, but are not limited to: anthrax, campylobacter,cholera, diphtheria, enterotoxigenic E. coli, giardia, gonococcus,Helicobacter pylori (Lee and Chen, 1994), Hemophilus influenza B,Hemophilus influenzanon-typable, meningococcus, pertussis, pneumococcus,salmonella, shigella, Streptococcus B, group A Streptococcus, tetanus,Vibrio cholerae, yersinia, Staphylococcus, Pseudomonas species andClostridia species.

Target viruses include, but are not limited to: adenovirus, dengueserotypes 1 to 4 (Delenda et al., 1994; Fonseca et al., 1994; Smucny etal., 1995), ebola (Jahrling et al., 1996), enterovirus, hepatitisserotypes A to E (Blum, 1995; Katkov, 1996;.Lieberman and Greenberg,1996; Mast, 1996; Shafara et al., 1995; Smedila et al., 1994; U.S. Pat.Nos. 5,314,808 and 5,436,126), herpes simplex virus 1 or 2, humanimmunodeficiency virus (Deprez et al., 1996), influenza, Japanese equineencephalitis, measles, Norwalk, papilloma virus, parvovirus B19, polio,rabies, rotavirus, rubella, rubeola, vaccinia, vaccinia constructscontaining genes coding for other antigens such as malaria antigens,varicella, and yellow fever.

Target parasites include, but are not limited to: Entamoeba histolytica(Zhang et al., 1995); Plasmodium (Bathurst et al., 1993; Chang et al.,1989, 1992, 1994; Fries et al., 1992a, 1992b; Herrington et al., 1991;Khusmith et al., 1991; Malik et al., 1991; Migliorini et al., 1993;Pessi et al., 1991; Tam, 1988; Vreden et al., 1991; White et al., 1993;Wiesmueller et al., 1991), Leishmania (Frankenburg et al., 1996),Toxoplasmosis, and the Helminths.

Antigens can also comprise those used in biological warfare such asricin, for which protection can be achieved via antibodies.

In this aspect and all other aspects described herein, a target antigenfor use in the compositions of the present invention can be expressed byrecombinant means, and in some embodiments, it can be expressed as afusion with an affinity or epitope tag (Summers and Smith, 1987;Goeddel, 1990; Ausubel et al., 1996), or with a third entity for thepurposes of forming a non-covalent linkage or complex with LFpolypeptide; chemical synthesis of an oligopeptide, either free orconjugated to carrier proteins, can be used to obtain antigen of theinvention (Bodanszky, 1993; Wisdom, 1994). Oligopeptides are considereda type of polypeptide.

In one embodiment, the target antigen is an intracellular pathogentarget antigen polypeptide. A pathogen has been defined as amicroorganism capable of causing damage to the host. An intracellularpathogen is a microorganism that can gain entry into the interior of acell, live inside host cells and cause damage to the host and/or hostcells. For example, the pathogen can be phagocytosed and/or endocytosedby a host cell, followed by the pathogen's escape from the phagosome orendosome. The pathogen then resides intracellularly to evadeother/subsequent host defense, such as antibodies, and to multiply.Phagocytosis by macrophages is a primary frontline host defensemechanism against pathogens. When a pathogen fails to escape from thephagosome or endosome, the phagocytosed or engulfed pathogen is digestedby the enzymes coming from the lysosomes. The digested, smaller peptidesderived from pathogen proteins are complexed with host cell MHCmolecules and displayed extracellularly to other immune cells in thehost so as to stimulate the immune system of the host to respond to thatparticular pathogen. An intracellular pathogen target antigenpolypeptide, when displayed and presented to host immune cells in thecontext of MHC molecules as described herein, can stimulate an immuneresponse in the host that can involve numerous cellular processes knownin the art of immunology. Aspects of such a response include an increasein cytokine production, increased antibody production, and increasedB-cell multiplication. Intracellular pathogens include but are notlimited to viruses, certain bacteria and certain protozoa. They cause arange of human diseases and ailments: tuberculosis, leprosy, typhoidfever, bacillary dysentery, plague, brucellosis, pneumonia, typhus;Rocky Mountain spotted fever, chlamydia, trachoma, gonorrhea,Listeriosis, scarlet/rheumatic fever, “strep” throat, hepatitis, AIDS,congenital viral infections, mononucleosis, Burkitts lymphoma and otherlymphoproliferative diseases, cold sores, genital herpes, genital warts,cervical cancer, leishmaniasis, malaria, and trypanosomiasis to name buta few.

In one embodiment, the target antigen polypeptide is an intracellularpathogen target antigen polypeptide from a prokaryotic pathogen. Aprokaryotic pathogen is a bacterium. In one embodiment, theintracellular prokaryotic pathogen includes but not limited toMyocobacterium tuberculosis, Mycobacterium leprae, Listeriamonocytogenes, Salmonella typhi, Shigella dysenteriae, Yersinia pestis,Brucella species, Legionella pneumophila, Rickettsiae, Chlamydia,Clostridium perfringens, Clostridium botulinum, Staphylococcus aureus,Treponema pallidum, Haemophilus influenzae, Treponema pallidum,Klebsiella pneumoniae, Pseudomonas aeruginosa, Cryptosporidium parvum,Streptococcus pneumoniae, Bordetella pertussis, and Neisseriameningitides.

In one embodiment, the target antigen polypeptide is an intracellularpathogen target antigen polypeptide from a viral pathogen, in which thevirus naturally infects mammalian host cells. In one embodiment, theviral pathogen includes but is not limited to Herpes simplex virustype-1, Herpes simplex virus type-2, HBV, Cytomegalovirus, Epstein-Barrvirus, Varicella-zoster virus, Human herpes virus 6, Human herpes virus7, Human herpes virus 8, Variola virus, Vesicular stomatitis virus,Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis Dvirus, Hepatitis E virus, poliovirus, Rhinovirus, Coronavirus, Influenzavirus A, Influenza virus B. Measles virus, Polyomavirus, HumanPapilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus,Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus,Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus,Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St.Louis Encephalitis virus, Murray Valley fever virus, West Nile virus,Rift Valley fever virus, Rotavirus A, Rotavirus B. Rotavirus C, Sindbisvirus, Rabies virus, Human T-cell Leukemia virus type-1, Hantavirus,Rubella virus and Simian Immunodeficiency virus.

In one embodiment, the target antigen polypeptide is an intracellularpathogen target antigen polypeptide of a parasitic pathogen. In oneembodiment, the intracellular parasitic pathogen includes but is notlimited to Myocobacterium tuberculosis, Mycobacterium leprae, Listeriamonocytogenes, Salmonella typhi, Shigella dysenteriae, Yersinia pestis,Brucella species, Legionella pneumophila, Rickettsiae, Chlamydia,Clostridium perfringens, Staphylococcus aureus, Treponema pallidum,Haemophilus influenzae, Treponema pallidum,Klebsiella pneumoniae,Pseudomonas aeruginosa, Streptococcus pneumoniae, Bordetella pertussis,Neisseria meningitides, Leishmania donovanii, Plasmodium species,Pneumocystis carinii, Trypanosoma species, Herpes simplex virus type-1,Herpes simplex virus type-2, HBV, Cytomegalovirus, Epstein-Barr virus,Varicella-zoster virus, Human herpes virus 6, Human herpes virus 7,Human herpes virus 8, Variola virus, Vesicular stomatitis virus,Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis Dvirus, Hepatitis E virus, poliovirus, Rhinovirus, Coronavirus, Influenzavirus A, Influenza virus B. Measles virus, Polyomavirus, HumanPapilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus,Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus,Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus,Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St.Louis Encephalitis virus, Murray Valley fever virus, West Nile virus,Rift Valley fever virus, Rotavirus A, Rotavirus B. Rotavirus C, Sindbisvirus, Rabies virus, Human T-cell Leukemia virus type-1, Hantavirus,Rubella virus and Simian Immunodeficiency virus.

In one embodiment, the target antigen polypeptide is an antigen of M.tuberculosis. In one embodiment, the target antigen polypeptide is aTB-specific antigen, including, but not limited to, TB1 (CFP)polypeptide comprising SEQ ID NO: 7 or a fragment thereof, oralternatively, TB2 (ESAT) polypeptide comprising SEQ ID NO: 8 or afragment thereof.

In another embodiment, a target antigen polypeptide is an intracellularpathogen target antigen polypeptide at least 15 amino acids long. Forexample, a target antigen can be the 91 amino acid fragment (amino acids27-117) of P. falciparum circumsporozoite protein, a predominant surfaceprotein, that is involved in invasion of liver cells by Plasmodiumsporozoites, which leads to malaria.

In alternative embodiments, the present invention can also be useful foreliciting a antigen-specific immune response against antigens such asviral antigens, such as sequestrin, to prevent the binding oferythrocytes to vascular endothelium in malaria by inducinganti-sequestrin antibodies. In alternative embodiments, the presentinvention can be used to elicit an antigen-specific immune responseagainst viral antigens such as for the induction of protectiveantibodies such as anti-hepatitis A, B or hepatitis E antibodies, usingthe whole inactivated virus, or alternatively virus-derived subunits orrecombinant products in the composition in combination with the LFn orfragment thereof.

In alternative embodiments, the compositions and methods as disclosedherein can be useful in the protection against tetanus, diphtheria andother toxin mediated diseases to induce the production of anti-toxinantibodies. For example, a tetanus “booster” is envisioned, where thecomposition as disclosed herein comprises LFn or a fragment thereof anda target antigen toxoid such as tetanus toxin or diphtheria, orfragments such as the tetanus C fragment. Boosting could be achievedfollowing primary immunization by injection or transcutaneousimmunization with the same or similar antigens, or woth standard vaccinecompostions, e.g., a toxoid vaccine.

Vaccination can als be used as a treatment for cancer and autoimmunedisease. For example, vaccination with a tumor antigen (e.g., prostatespecific antigen or PSA) can induce an immune response in the form ofantibodies, CTLs and lymphocyte proliferation which allows the body'simmune system to recognize and kill tumor cells. Targeting dendriticcells, of which Langerhans cells are a specific subset, has been shownto be an important strategy in cancer immunotherapy.

In one embodiment of this aspect and all other aspects described herein,a target antigen is a tumor antigen. Many tumors are associated with theexpression of a particular protein and/or the over-expression of certainproteins. For example, prostate cancer is associated with elevatedlevels of protein such as Prostate Specific Antigen (PSA). Breastcancers can be associated with the expression and/or over-expression ofprotein such as Her-2, Muc-1, CEA, etc. Thus, considerable attention hasbeen aimed at trying to generate immune responses, particularlydeveloping CMI, to such antigens in the treatment of such malignancies.Tumor antigens useful in this aspect and all other aspects describedherein, include, for example PSA, Her-2, Mic-1 and CEA. Other tumorantigens include those epitopes which are recognized in eliciting T cellresponses, including but not limited to the following: prostate cancerantigens (such as PSA, PSMA, etc.), breast cancer antigens (such asHER2/neu, mini-MUC, MUC-1, HER2 receptor, mammoglobulin, labyrinthine,SCP-1, NY-ESO-1, SSX-2, N-terminal blocked soluble cytokeratin, 43 kDhuman cancer antigens, PRAT, TUAN, Lb antigen, carcinoembryonic antigen,polyadenylate polymerase, p53, mdm-2, p21, CA15-3, oncoprotein18/stathmin, and human glandular kallikrein), melanoma antigens, and thelike. Tumor antigens useful to be delivered by an LF polypeptideaccording to the methods and compositions herein are described in usefor melanoma (U.S. Pat. Nos. 5,102,663, 5,141,742, and 5,262,177 whichare incorporated herein in their entirety by reference), prostatecarcinoma (U.S. Pat. No. 5,538,866), and lymphoma (U.S. Pat. Nos.4,816,249, 5,068,177, and 5,227,159 which are all incorporated herein intheir entirety by reference).

In one embodiment of this aspect and all other aspects described herein,a target antigen is a T-cell receptor oligopeptide. For example,vaccination with T-cell receptor oligopeptide can induce an immuneresponse that halts progression of autoimmune disease (U.S. Pat. Nos.5,612,035 and 5,614,192; Antel et al, 1996; Vandenbark et al, 1996).U.S. Pat. No. 5,552,300 (which is incorporated herein in its entirety byreference) also describes antigens suitable for treating autoimmunedisease. In one embodiment of this aspect and all other aspectsdescribed herein, a target antigen is a T-cell receptor V antigen, whichis discussed in U.S. Pat. No. 5,552,300 (which is incorporated herein inits entirety by reference).

In one embodiment of this aspect and all other aspects described herein,a target antigen is a misfolded or improperly folded protein orpolypeptide, for example a protein which is not in its nativeconformation. Examples of such proteins include proteins which areexpressed by a mutant nucleic acid sequence which encodes for theprotein. Typically, if a mutation in the nucleic acid results in a nonconservative amino acid change in the protein relative to the wild-type(i.e. non-mutant nucleic acid) the protein can have a conformationchange (i.e. not be in its native conformation) or misfolded. In someinstances, misfolded polypeptides due to a non-conservative amino acidchange can result in what is commonly known in the art as a“gain-of-function” where the misfolded polypeptide has a biologicalfunction different and/or in addition to the correctly foldedpolypeptide. The term a “gain of function mutation” refers to a geneticmutation where the mutant gene produces a protein which has extracapabilities relative to the wild type. Such different functions orextra capabilities, can be, for example, protein aggregation, abnormalsubcellular localization, and other functions which do not occur withcorrectly folded (i.e. the native conformation) polypeptides. Examplesare, for instance, amyloid proteins, as well as mutated proteins inneurodegenerative diseases, including but not limited to the mutatedSOD1 or mutated TDP-43 genes in ALS, mutated genes which produceproteins in polyglutamine disorders, such as mutated huntingtin gene inHuntington's Disease and mutated beta-amyloid in Alzheimer's disease.

Without wishing to be bound by theory, Amyloids are insoluble fibrousprotein aggregates sharing specific structural traits. Abnormalaccumulation of amyloid in organs can lead to amyloidosis, and can playa role in various other neurodegenerative diseases. Diseases withamyloids include, but are not limited to Alzheimer's disease (Betaamyloid), Type 2 diabetes mellitus (IAPP), Parkinson's disease(Alpha-synuclein), Transmissible spongiform encephalopathy aka “Mad CowDisease” (Prion), Huntington's Disease (Polyglutamine repeats),Medullary carcinoma of the thyroid (Calcitonin), Amyloidosis(Immunoglobulin), Sporadic Inclusion Body Myositis (S-IBM),pheochromocytoma and Osteomyelitis. Abnormal protein aggregation is alsopresent in many neurodegenerative disorders, including AD, Parkinson'sdisease, Creutzfeldt-Jakob disease, motor neuron diseases such as ALS, alarge group of polyglutamine disorders, including Huntington's disease,as well as diseases of peripheral tissue like familial amyloidpolyneuropathy (FAP).

In one embodiment of this aspect and all other aspects described herein,a target antigen can be a protein involved in a disease or disorder. Asused herein, a gene “involved” in a disease or disorder includes a gene,the normal or aberrant expression or function of which effects or causesthe disease or disorder or at least one symptom of said disease ordisorder. The term “gain-of-function mutation” as used herein, refers toany mutation in a gene in which the protein encoded by said gene (i e.,the mutant protein) acquires a function not normally associated with theprotein (i. e., the wild type protein) and causes or contributes to adisease or disorder. The gain-of-function mutation can be a deletion,addition, or substitution of a nucleotide or nucleotides in the genewhich gives rise to the change in the function of the encoded protein.In one embodiment, the gain-of-function mutation changes the function ofthe mutant protein or causes interactions with other proteins. Inanother embodiment, the gain-of-function mutation causes a decrease inor removal of normal wild-type protein, for example, by interaction ofthe altered, mutant protein with said normal, wild-type protein.

In one embodiment of this aspect and all other aspects described herein,a target antigen can be a protein expressed from a gene with apolymorphism. The term “polymorphism” as used herein, refers to avariation (e. g. , a deletion, insertion, or substitution) in a genesequence that is identified or detected when the same gene sequence fromdifferent source subjects (but from the same organism) are compared. Forexample, a polymorphism can be identified when the same gene sequencefrom different subjects (but from the same organism) are compared.Identification of such polymorphisms is routine in the art, themethodologies being similar to those used to detect, for example, breastcancer point mutations. Identification can be made, for example, fromDNA extracted from a subject's lymphocytes, followed by amplification ofpolymorphic regions using specific primers to said polymorphic region.Alternatively, the polymorphism can be identified when two alleles ofthe same gene are compared. A variation in sequence between two allelesof the same gene within an organism is referred to herein as an “allelicpolymorphism”. The polymorphism can be at a nucleotide within a codingregion but, due to the degeneracy of the genetic code, no change inamino acid sequence is encoded. Alternatively, polymorphic sequences canencode a different amino acid at a particular position, but the changein the amino acid does not affect protein function. Polymorphic regionscan also be found in non-encoding regions of the gene.

In one embodiment of this aspect and all other aspects described herein,a target antigen can be a protein with a polyglutamine domain, or anexpanded polyglutamine domain. The term “polyglutamine domain” as usedherein, refers to a segment or domain of a protein that consist of aconsecutive glutamine residues linked to peptide bonds. In oneembodiment the consecutive region includes at least 5 glutamineresidues. The term “expanded polyglutamine domain” or “expandedpolyglutamine segment”, as used herein, refers to a segment or domain ofa protein that includes at least 35 consecutive glutamine residueslinked by peptide bonds. Such expanded segments are found in subjectsafflicted with a polyglutamine disorder, as described herein, whether ornot the subject has shown to manifest symptoms. The term “trinucleotiderepeat” or “trinucleotide repeat region” as used herein, refers to asegment of a nucleic acid sequence e. g.) that consists of consecutiverepeats of a particular trinucleotide sequence. In one embodiment, thetrinucleotide repeat includes at least 5 consecutive trinucleotidesequences. Exemplary trinucleotide sequences include, but are notlimited to, CAG, CGG, GCC, GAA, CTG, and/or CGG.

In one embodiment of this aspect and all other aspects described herein,a target antigen can be a protein expressed in a trinucleotide repeatdisease. The term “trinucleotide repeat diseases” as used herein, refersto any disease or disorder characterized by an expanded trinucleotiderepeat region located within a gene, the expanded trinucleotide repeatregion being causative of the disease or disorder. Examples oftrinucleotide repeat diseases include, but are not limited tospino-cerebellar ataxia type 12 spino-cerebellar ataxia type 8, fragileX syndrome, fragile XE Mental Retardation and myotonic dystrophy.Preferred trinucleotide repeat diseases for treatment according to thepresent invention are those characterized or caused by an expandedtrinucleotide repeat region at the 5′end of the coding region of a gene,the gene encoding a mutant protein which causes or is causative of thedisease or disorder.

In one embodiment of this aspect and all other aspects described herein,a target antigen is can be a protein expressed in a polyglutaminedisorder. The term “polyglutamine disorder” as used herein, refers toany disease or disorder characterized by an expanded of a (CAG) nrepeats at the 5′end of the coding region (thus encoding an expandedpolyglutamine region in the encoded protein). In one embodiment,polyglutamine disorders are characterized by a progressive degenerationof nerve cells. Examples of polyglutamine disorders include but are notlimited to: Huntington's disease, spino-cerebellar ataxia type 1,spino-cerebellar ataxia type 2, spino-cerebellar ataxia type 3 (alsoknow as Machado-Joseph disease), and spino-cerebellar ataxia type 6,spino-cerebellar ataxia type 7 and dentatorubral-pallidoluysian atrophy.

In one embodiment, a target antigen polypeptide is folded in its nativeconformation. In one embodiment, a target antigen polypeptide is part ofa multi-molecular polypeptide complex. In one embodiment, a targetantigen polypeptide is a subunit polypeptide of a multi-molecularpolypeptide target antigen.

In some embodiments, a target antigen can be an intact (i.e. an entireor whole or complete) target antigen which is delivered to the cytosolof a cell by a non-linked or non-covalently linked LF polypeptide asdescribed herein. By “intact” in this context is meant that the targetantigen is the full length target antigen as that antigen polypeptideoccurs in nature. This is in direct contrast to delivery of only a smallportion or peptide of the target antigen. By delivering an intact targetantigen to a cell, the LFn polypeptide enables or facilitates thetranslocation of the whole target antigen across the cell membrane andthe display of a full range of epitopes of the intact target antigen incomplexes with MHC I molecules. Moreover, this also facilitatesdetection of a cell mediated immune (CMI) response to a full range ofepitopes of the intact target antigen, rather than just a single orselected few peptide epitopes. CMI occurs when T cells (lymphocytes)bind to the surface of other cells that display the antigen and triggera response, e. g. production and release of cytokines. The response caninvolve other lymphocytes and any of the other white blood cells(leukocytes).

Accordingly, the use of an LFn polypeptide (which is non-linked ornon-covalently linked to the intact antigen) in the methods andcompositions described herein results in a stronger and more robust CMIresponse to the intact target antigen as compared to use of the intacttarget antigen alone or to a part (i.e. a peptide) of the targetantigen, as a CMI response can be raised against essentially any epitopeof the whole antigen.

In some embodiments, the intact target antigen can be divided intofragments, or parts, of the whole target antigen, for example, at leasttwo, or at least 3, or at least 4, or a least 5 or more target antigenfragments, depending on size of the intact target antigen protein. Thesefragments of the whole target antigen can be used, for example, as aquality control to filter out false positives of a positive CMIresponse. By way of an example only, a positive CMI response to a wholetarget antigen can be confirmed by assessing a CMI response to a panelof target antigens which are fragments of the whole target antigen. Atrue CMI response is confirmed if one or two of the fragments give apositive response, but not all fragments. If a positive CMI response isdetected for all fragments, it is likely that the positive CMI responsewas a false positive.

In some embodiments, an intact target antigen can be divided into manyparts, depending on the size of the initial target antigen, for use as apanel of sub-target antigens. Typically, where a whole target antigen isa multimer polypeptide, the whole target protein can be divided intosub-units and/or domains which can each individually can be mixed withan LF polypeptide and used in assay methods and compositions asdisclosed herein. Alternatively, an intact target antigen can be dividedinto fragments, or parts of the whole target antigen, for example, atleast two, or at least 3, or at least 4, or at least 5, or at least 6,or at least 7, or at least 8, or at least about 9, or at least about 10,or at least about 11, or at least about 12, or at least about 13, or atleast about 15, or at least about 20, or at least about 25, or more than25 fragments, and each fragment, individually or in combination, mixedwith an LF polypeptide for use in assay methods and compositions asdisclosed herein.

The fragmentation or division of a full length target antigenpolypeptide can be an equal division of the full length target antigenpolypeptide, or alternatively, in some embodiments, the fragmentation isasymmetrical or unequal. As a non-limiting example, where a targetantigen is divided into two overlapping fragments, a target antigen canbe divided into fragments of approximately the same (equal) size, oralternatively one fragment can be about 45% of the whole target antigenand the other fragment can be about 65%. As further non-limitingexamples, a whole target antigen can be divided into a combination ofdifferently sized fragments, for example, where a target antigen isdivided into two fragments, fragments can be divided into about 40% andabout 70%, or about 45% and about 65%; or about 35% and about 75%; orabout 25% and about 85% of the whole target antigen. Any combination ofoverlapping fragments of a full length whole target antigen isencompassed for use in the generation of a panel of target antigens. Asan illustrative example only, where a target antigen is divided into 5portions, the portions can be divided equally (i.e. each overlappingfragment is about 21 to 25% of the entire full length if the targetantigen) or unequally (i.e. a target antigen can be divided into thefollowing 5 overlapping fragments; fragment 1 is about 25%, fragment 2is about 5%, fragment 3 is about 35%, fragment 4 is about 10% andfragment 5 is about 25% of the size of the full length target antigen,provided each fragment overlaps with at least one other fragment).

A target antigen which is a peptide, (i.e. a lengths of anywhere between6 residues to 20 residues) can be delivered by a non-linked LFpolypeptide. Polypeptides can also by synthesized as branched structuressuch as those disclosed in U.S. Pat. Nos. 5,229,490 and 5,390,111 whichare incorporated herein by reference. Antigenic polypeptides include,for example, synthetic or recombinant B-cell and T-cell epitopes,universal T-cell epitopes, and mixed T-cell epitopes from one organismor disease and B-cell epitopes from another.

As noted above, target antigen can be obtained through recombinant meansor peptide synthesis. Other sources include, natural sources orextracts. In any event, the antigen can be purified by means of theantigen's physical and chemical characteristics, preferably byfractionation or chromatography (Janson and Ryden, 1989; Deutscher,1990; Scopes, 1993).

A multivalent target antigen formulation can be used to induce an immuneresponse to more than one antigen at the same time. Conjugates can beused to induce an immune response to multiple antigens, to boost theimmune response, or both. Additionally, toxins can be boosted by the useof toxoids, or toxoids boosted by the use of toxins.

In some embodiments, a target antigen can be solubilized in water, asolvent such as methanol, or a buffer. Suitable buffers include, but arenot limited to, phosphate buffered saline Ca ²⁺/Mg²⁺ free (PBS), normalsaline (150 mM NaCl in water), and Tris buffer. Target antigen notsoluble in neutral buffer can be solubilized in 10 mM acetic acid andthen diluted to the desired volume with a neutral buffer such as PBS. Inthe case of antigen soluble only at acid pH, acetate-PBS at acid pH canbe used as a diluent after solubilization in dilute acetic acid.Glycerol can be a suitable non-aqueous buffer for use in the presentinvention.

D. Additional Moieties and Adjuvants

In some embodiments, adjuvants in addition to the LF polypeptide can beincluded in the compositions as disclosed herein that comprise an LFpolypeptide, such as an LFn polypeptide and a target antigen which isnot covalently linked to the LFn polypeptide.

Adjuvants are a heterogeneous group of substances that enhance theimmunological response against an antigen that is administeredsimultaneously. In some instances, adjuvants are added to a vaccine toimprove the immune response so that less vaccine is needed. Adjuvantsserve to bring the antigen—the substance that stimulates the specificprotective immune response—into contact with the immune system andinfluence the type of immunity produced, as well as the quality of theimmune response (magnitude or duration). Adjuvants can also decrease thetoxicity of certain antigens; and provide solubility to some vaccinecomponents. Almost all adjuvants used today for enhancement of theimmune response against antigens are particles or form particlestogether with the antigen. In the book “Vaccine Design—the subunit andadjuvant approach” (Ed: Powell & Newman, Plenum Press, 1995) almost allknown adjuvants are described both regarding their immunologicalactivity and regarding their chemical characteristics. The type ofadjuvants that do not form particles are a group of substances that actas immunological signal substances and that under normal conditionsconsist of the substances that are formed by the immune system as aconsequence of the immunological activation after administration ofparticulate adjuvant systems.

Typically adjuvants are particulate systems where the antigens areassociated or mixed with or to a matrix, which has the characteristicsof being slowly biodegradable. Adjuvants which are matrix systems shouldnot degrade to form toxic metabolites. The main kinds of matrices thatcan be used are mainly substances originating from a body, including,for example lactic acid polymers, poly-amino acids (proteins),carbohydrates, lipids and biocompatible polymers with low toxicity.Combinations of these groups of substances originating from a body orcombinations of substances originating from a body and biocompatiblepolymers can also be used. Lipids are the preferred substances sincethey display structures that make them biodegradable as well as the factthat they are a critical element in all biological membranes.

Adjuvants for vaccines are well known in the art. Suitable additionaladjuvants include, but are not limited to: complete Freund's adjuvant,incomplete Freund's adjuvant, saponin, mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyaninons, peptides, oil or hydrocarbon emulsions, keyholelimpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Selection of an adjuvant depends on the animal subject to bevaccinated. Additional examples include, but are not limited to,monoglycerides and fatty acids(e. g. a mixture of mono-olein, oleicacid, and soybean oil); mineral salts, e.g., aluminium hydroxide andaluminium or calcium phosphate gels; oil emulsions and surfactant basedformulations, e.g., MF59 (microfluidised detergent stabilisedoil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2](oil-in-water emulsion+MPL+QS-21), Montanide ISA-51 and ISA-720(stabilised water-in-oil emulsion); particulate adjuvants, e.g.,virosomes (unilamellar liposomal vehicles incorporating influenzahaemagglutinin), ASO4 ([SBAS4] Al salt with MPL), ISCOMS (structuredcomplex of saponins and lipids), polylactide co-glycolide (PLG);microbial derivatives (natural and synthetic), e.g., monophosphoryllipid A (MPL), Detox (MPL+M. Phlei cell wall skeleton), AGP [RC-529](synthetic acylated monosaccharide), DC_Chol (lipoidal immunostimulatorsable to self organize into liposomes), OM-174 (lipid A derivative), CpGmotifs (synthetic oligonucleotides containing immunostimulatory CpGmotifs), modified LT and CT (genetically modified bacterial toxins toprovide non-toxic adjuvant effects); endogenous human immunomodulators,e.g., hGM-CSF or hIL-12 (cytokines that can be administered either asprotein or plasmid encoded), Immudaptin (C3d tandem array) and inertvehicles, such as gold particles. Newer adjuvants are described in U.S.Pat. No. 6,890,540, United States Patent Application No. 20050244420,and PCT/SE97/01003, the contents of which are incorporated herein byreference in their entirety. The adjuvant can also be selected from thegroup consisting of QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G,CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF,B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59.

It is preferred that any additional adjuvants be a pharmaceuticallyacceptable adjuvant. For example, oils or hydrocarbon emulsion adjuvantsshould not be used for human vaccination. One example of an adjuvantsuitable for use with humans is alum (alumina gel). Details of commonadjuvants which are contemplated to be added to the compositions of thepresent invention are discussed below:

Complete Freund's Adjuvant (CFA): A mineral oil adjuvant; uses awater-in-oil emulsion which is primarily oil. For many years theadjuvant of choice was complete Freund's adjuvant. This adjuvant, whilepotent immunogenically, also has had a significant history of frequentlyproducing abscesses, granulomas and tissue sloughs. It contains paraffinoil, killed mycobacteria and mannide monoosleate. The paraffin oil isnot metabolized; it is either expressed through the skin (via agranuloma or abscess) or phagocytized by macrophages. Multiple exposuresto CFA will cause severe hypersensitivity reactions. Accidental exposureof personnel to CFA can result in sensitization to tuberculin.

Incomplete Freund's Adjuvant (IFA): Also a mineral oil adjuvant.Composition similar to CFA but does not contain the killed mycobacteriaso does not produce as severe reactions. Used for the boosterimmunizations following the initial injection with antigen-CFA. IFA canbe used for initial injection if the antigen is strongly immunogenic.

Montanide ISA (Incomplete Seppic Adjuvant): A mineral oil adjuvant. Usesmannide oleate as the major surfactant component. The antibody responseis generally similar to that with IFA. Montanide ISA may have a lessenedinflammatory response.

Ribi Adjuvant System (RAS): An oil-in-water emulsion that containsdetoxified endotoxin and mycobacterial cell wall components in 2%squalene. Multiple formulations are commercially available, dependent onuse. Is an alternative to CFA. Lower viscosity than CFA. Results(titers) often comparable to those with CFA. The squalene oil ismetabolizable. RAS has a lower incidence of toxic reactions.

TiterMax: Another water-in-oil emulsion, this preperation combines asynthetic adjuvant and microparticulate silica with the metabolizableoil squalene. The copolymer is the immunomodulator component. Antigen isbound to the copolymer and presented to the immune cells in a highlyconcentrated form. Less toxicity than CFA. TiterMax usually produces thesame results as CFA.

Syntex Adjuvant Formulation (SAF): A preformed oil-in-water emulsion.Uses a block copolymer for a surfactant. A muramyl dipeptide derivativeis the immunostimulatory component. All in squalene, a metabolizableoil. SAF can bias the humoral response to IgG2a in the mouse, but isless toxic than CFA.

Aluminum Salt Adjuvants: Most frequently used as adjuvants for vaccineantigen delivery. Generally weaker adjuvants than emulsion adjuvants.Aluminum Salt Adjuvants are best used with strongly immunogenicantigens, but result generally in mild inflammatory reactions.

Nitrocellulose-adsorbed antigen: The nitrocellulose is basically inert,leading to almost no inflammatory response. Slow degradation ofnitrocellulose paper allows prolonged release of antigen. Does notproduce as dramatic an antibody response as CFA. Nitrocellulose-adsorbedantigen is good for use if only a small amount of antigen can berecovered from a gel band, e.g., for animal immunization.

Encapsulated or entrapped antigens: Permits prolonged release of antigenover time; can also have immunostimulators in preparation for prolongedrelease. Preparation of encapsulated or entrapped antigens is complex.

Immune-stimulating complexes (ISCOMs): Antigen modifiedsaponin/cholesterol micelles. Stable structures are formed which rapidlymigrate to draining lymph nodes. Both cell-mediated and humoral immuneresponses are achieved. Low toxicity; ISCOMs can elicit significantantibody response. Quil A is one example, QS-21 is another.

GerbuR adjuvant: An aqueous phase adjuvant which uses immunostimulatorsin combination with zinc proline. GerbuR does not have a depot effectand has minimal inflammatory effect. GerbuR requires frequent boostingto maintain high titers.

Alum is a preferred adjuvant. Another group of adjuvants include immunestimulators such as cytokines IL-12, IL-4 and costimulatory moleculessuch as B7. A wide range of molecules having immune stimulating effectsare known including accessory molecules such as ICAM and LFA. In apreferred embodiment GM-CSF is administered to the patient before theinitial immune administration. GM-CSF can be administered using a viralvector or an isolated protein in a pharmaceutical formulation.Combinations of adjuvants can be used such as CM-CSF, I CAM and LFA.While a strong immune response is typically generated to infectiousdisease antigens, tumor associated antigens typically generate a weakerimmune response. Thus, immune stimulators such as described above arepreferably used with them.

E. Methods to Assay for a CTL Response

In one embodiment of this aspect and all other aspects described herein,the delivery to a cell of a non-linked or non-covalently linked targetantigen by an LF polypeptide can be assessed by measuring a CMI responseto the target antigen. CMI assays are known in the art and described,for example, in United States Patent Application 20050014205,WO/1987/005400, U.S. Pat. No. 5,674,698 and commercially available kitssuch as IMMUNKNOW® CYLEX Immune cell function assay Product No. 4400,which are incorporated in their entirety by reference herein for use inthe present invention.

F. Applications of an LFN Polypeptide with a Non-Linked orNon-Covalently Linked Target Antigen

The immunogenic compositions of the present invention can be used toelicit a specific-immune response against the target antigen. In analternative embodiment, an LF polypeptide, such as an LFn polypeptide orfragment thereof can be used to stabilize a target antigen which isunstable, without the need for being bound to the target antigen. Forexample, certain target antigens are difficult to express and/or areunstable and thus fold incorrectly to form a misfolded protein comparedto its native active conformation. For such a protein, while still ableto elicit a CMI response, the response will likely only be responsive tothe improperly folded protein, and can not render any therapeutic and/orprophylactic benefit to the subject in which a CMI response is raised.While not wishing to be bound by theory, it is hypothesized that thepresence of an LF polypeptide such as a polypeptide corresponding to SEQID NO: 3 or SEQ ID NO: 4 can stabilize certain such unstable targetantigen proteins. The inventors have discovered that a fragment of LFnwhich is about at least 250 amino acids or less, or about at least 150amino acids or less, or about at least 103 amino acids or less, or aboutat least 80 amino acids or less is able to stabilize unstable targetantigens without needing to be fused to such target antigens. It ispossible under this model that co-expression of non-linked LFpolypeptide and target antigen can be benefical during the expression ofthe target antigen, or immediately after expression of the targetantigen.

In some embodiments, the compositions as disclosed herein can be used togenerate an immune response against the target antigen, such as forexample use as a vaccine. An exemplary composition is a therapeuticallyeffective amount of an LFn polypeptide (corresponding to SEQ ID NO: 3 or4) or a fragment, homologue or variant thereof and a non-linked ornon-covalently linked target antigen to induce an immune reaction. Thecomposition acts as a prophylactic immunogen, optionally included in apharmaceutically-acceptable and compatible carrier.

The term “pharmaceutically-acceptable and compatible carrier” as usedherein, and described more fully below, includes (i) one or morecompatible solid or liquid filler diluents or encapsulating substancesthat are suitable for administration to a human or other animal, and/or(ii) a system, capable of delivering the molecule to a target cell. Inthe present invention, the term “carrier” thus denotes an organic orinorganic ingredient, natural or synthetic, with which the molecules ofthe invention are combined to facilitate application. The term“therapeutically-effective amount” is that amount of the presentpharmaceutical composition which produces a desired result or exerts adesired influence on the particular condition being treated. Forexample, the amount necessary to raise an immune reaction to provideprophylactic protection. Typically when the composition is being used asa prophylactic immunogen at least one “boost” will be administered at aperiodic interval after the initial administration. Variousconcentrations can be used in preparing compositions incorporating thesame ingredient to provide for variations in the age of the patient tobe treated, the severity of the condition, the duration of the treatmentand the mode of administration.

In one embodiment, the compositions as disclosed herein is amulticomponent vaccine which can comprise other immunogenicpolypeptides, such as other adjuvants in addition to an LF polypeptidesuch as LFn or fragments thereof. A multicomponent vaccine can containadditional adjuvant(s) to elicit T cell responses, as well as otherantigens and/or adjuvants to elicit B cell responses.

In some embodiments, the compositions are administered to a subject by amethod of immunization, and in some embodiments, the method ofimmunization involves multiple administration regimens; e.g., a firstadministration to prime where the LFn can be used to deliver anon-linked or non-covalently linked target antigen; and then a secondadministration to “boost” the CMI using the LFn and a different novelnon-linked or non-covalently linked target antigen.

In some embodiments, one can also use a composition comprising acocktail of different LFn and non-linked or non-covalently linked targetantigens to prime and boost with either a variety of different targetantigens or with LFn in the presence of multiple target antigens.

In one embodiment of this aspect and all other aspects described herein,a pharmaceutical composition comprising an LF polypeptide and a targetantigen can be used to generate a range of T cells that recognize andinteract with a diverse range of antigens, for example, from differentHIV strains. In some embodiments, the DNA sequence encoding the LFpolypeptide and target antigen can also be used as a DNA-based vaccine.In trying to generate an immune reaction such as with a vaccinecomposition as disclosed herein, an adjuvant can also be used.

The immune stimulatory composition of the present invention can be usedadvantageously with other treatment regimens. For example, the systemcan be used in conjunction with traditional treatment options for cancerincluding surgery, radiation therapy, chemotherapy and hormone therapy.For example, a breast cancer vaccine (i.e. a composition) comprising anLF polypeptide and a non-linked or non-covalently linked target antigencan be used in conjunction with tamoxifen citrate, which interferes withthe activity of estrogen. The system can also be combined withimmunotherapy, e.g. using HERCEPTIN™ (trastuzumab), an anti-HER2humanized monoclonal antibody developed to block the HER2 receptor; bonemarrow transplantation; and peripheral blood stem cell therapy can alsobe used. Other preferred treatment regimens that can be used inconjunction with the compositions described herein include angiogenesisinhibitors and cytotoxic agents.

The term “compatible”, as used herein, means that the components of thepharmaceutical compositions are capable of being commingled with eachother, in a manner that does not substantially impair the desiredpharmaceutical efficacy.

In one embodiment, the vaccine composition comprises an LF polypeptidesuch as LFn and a non-linked or non-covalently linked target antigenwhich is expressed and purified from insect cells. In one embodiment,the vaccine composition comprises a plurality of LF polypeptides such asLFn and a plurality of non-linked or non-covalently linked targetantigens that are expressed and purified from insect cells, wherein thetarget antigen polypeptides are different but all are from a singleintracellular pathogen. In one embodiment, the plurality of targetantigen polypeptides are all from a single polypeptide from a singleintracellular pathogen. In one embodiment, the vaccine compositioncomprises a plurality of LF polypeptides and a plurality of non-linkedor non-covalently linked target antigens. In some embodiments, an LFpolypeptide and a plurality of non-linked target antigens are expressedand purified from insect cells, wherein each target antigen polypeptideis different but all are from several intracellular pathogens. Forexample, a vaccine composition for raising a cell-mediated immune (CMI)response to mumps, measles and rubella viruses can have at least threedifferent non-linked or non-covalently linked target antigens, eachspecific to mumps, measles and rubella viruses, respectively.

In another embodiment, the vaccine composition comprises an LFpolypeptide such as LFn and a non-covalently linked target antigen,wherein the LFn polypeptide is N-glycosylated. The N-glycosylation canbe at asparagine 62, 212 and/or 286 relative to the LFn of SEQ ID NO: 3or SEQ ID NO: 4.

In some embodiments, the vaccine composition described herein furthercomprises pharmaceutical excipients including, but not limited tobiocompatible oils, physiological saline solutions, preservatives,osmotic pressure controlling agents, carrier gases, pH-controllingagents, organic solvents, hydrophobic agents, enzyme inhibitors, waterabsorbing polymers, surfactants, absorption promoters and anti-oxidativeagents.

In one embodiment, the invention provides a composition comprising an LFpolypeptide such as LFn and a non-linked or non-covalently linked targetantigen as described herein and an isolated mammalian cell. The isolatedcell is preferably capable of processing and presenting target antigenfragments for display with MHC molecules. Such antigen-presenting cellscan express MHC class I molecules only (so-called “non-professional”antigen presenting cells (APC'S)), or MHC Class I and class II molecules(so-called “professional” APC'S). Thus, in one embodiment, the mammaliancell is an antigen presenting cell, including a professional APC and/ora non-professional APC. Professional APC's include, e. g., macrophages,dendritic cells and B cells. In one embodiment, an LF polypeptide suchas LFn and/or a non-linked or non-covalently linked target antigen canbe expressed and purified from insect cells. In one embodiment, themammalian cells are isolated from a subject who can have been exposed toa pathogen. Such a composition is useful in screening for exposure topathogens, such as a CMI response assay or for a mass vaccinationprogram. CMI assays are known in the art, for example, in United StatesPatent Application 20050014205, WO/1987/005400, U.S. Pat. No. 5,674,698and commercially available kits such as IMMUNKNOW® CYLEX Immune cellfunction assay Product No. 4400.

G. Kits

Another aspect of the present invention relates to kits for producing acomposition which is useful for eliciting a CMI against a desired targetantigen, when the target antigen is not covalently linked to the LFnpolypeptide. Such kits can be prepared from readily available materialsand reagents. For example, such kits can comprise any one or more of thefollowing materials: biologically active LFn, reaction tubes,instructions for testing LFn activity and reagents for addition of theuser's preferred target antigen. A wide variety of kits and componentscan be prepared according to the present invention, depending upon theintended use of the kit, the particular target antigen and the needs ofthe user.

In an embodiment of such an aspect and all other aspects describedherein, a kit comprises an LF polypeptide such as LFn as describedherein and packaging materials therefor. In some embodiments, the kitalso comprises a non-linked or non-covalently linked target antigen.Such a kit is useful in screening for exposure to pathogens, such as ina CMI response assay, or for a mass vaccination program. Packagingmaterials can include, but are not limited to adjuvants, diluents,alcohols wipes for disinfecting the site of injection, disposable fixvolume syringes, dosage chart and immunization schedule.

In an embodiment of such an aspect and all other aspects describedherein, a kit described herein comprises a plurality of LF polypeptidesand a plurality of non-covalently linked target antigens. In oneembodiment, individual members of the target antigens comprise differentantigen polypeptides to a different portion of the same target antigenpolypeptide.

A CMI assay is important for assessing both the exposure of a subject toa target antigen and a subject's ability to respond to an infection by apathogenic agent such as a microorganism, virus or parasite, to mount anautoimmune response such as in diabetes or to protect against cancers orother oncological conditions. Consequently, reference to “measuring aCMI response to a target antigen in a subject” encompasses immunediagnosis of infectious and autoimmune diseases, a marker forimmunocompetence and the detection of T-cell responses to endogenousand/or exogenous antigens (including a measure of the efficacy of avaccine) as well as a marker for inflammatory diseases and cancer.Monitoring CMI pre- and post-transplantation is necessary in themanagement of organ transplant patients. A CMI assay can also be used totitrate initial immunosuppression reduction and its subsequent increasein these patients.

As discussed above, any of a range of target antigens can be tested suchas those specific for a particular organism, pathogen, virus,auto-antigen or cancer cell. Alternatively, more general agents can beused to test generic capacity to mount a cell-mediated immune response.Examples of the latter include skin tests (e.g., PPD) from M.tuberculosis and tetanus toxoid. In general, however, any peptide,polypeptide or protein, glycoprotein, phosphoprotein, phospholipoproteincan be included in a non-linked or non-covalently linked from with an LFpolypeptide in the compostion as described herein. These includeantigens from pathogens, particularly, but not necessarily intracellularpathogens. The pathogens include, for example, any of the viral,bacterial, fungal or parasitic pathogens described herein, among others.The antigen can also include tumor antigens and/or autoimmune antigens.

H. Systems

In one aspect, provided herein is a system for measuring a cell mediatedimmune response (CMI) to a target antigen in a subject, the systemcomprising a computer processor and a computer-readable physical storagemedium having instructions recorded thereon sufficient to implement aprocess, employing the computer processor, for measuring a cell-mediatedimmune response, the instructions for said process comprising:

-   -   a) instructions for receiving data regarding the level of at        least one cytokine released in a biological sample in response        to contacting a cell in said sample with at least one fusion        polypeptide comprising a portion of an LF polypeptide lacking LF        enzymatic activity but sufficient to promote transmembrane        delivery of said fusion polypeptide to a cell, said portion        fused to a target antigen polypeptide or to a fragment thereof,        wherein said contacting permits transmembrane delivery of said        target antigen to a said cell, which cell processes and displays        at least one epitope of said antigen on its surface; and    -   b) instructions for comparing the level of said at least one        cytokine in said biological sample with a reference level of        said at least one cytokine,    -   c) instructions for transmitting to a user interface a result of        said comparison, wherein an increase in the level of said at        least one cytokine in said biological sample from the subject as        compared to a reference level indicates a cell mediated immune        response (CMI) to the target antigen in the subject.

In another aspect, provided herein is a computer-readable physicalstorage medium having instructions recorded thereon sufficient toimplement a process, employing a computer processor, for measuring acell-mediated immune response, the instructions for said processcomprising:

-   -   a) instructions for receiving data regarding the level of at        least one cytokine released in a biological sample, said data        obtained by:        -   i) incubating a biological sample from said subject with at            least one fusion polypeptide, the fusion polypeptide            comprising a portion of an LF polypeptide lacking LF            enzymatic activity but sufficient to promote transmembrane            delivery of said fusion polypeptide to a cell, said portion            fused to a target antigen polypeptide or to a fragment            thereof, wherein said biological sample comprises cells of            the immune system that release at least one cytokine in            response to an antigen and wherein said incubating permits            transmembrane delivery of said target antigen to a cell,            which cell processes and displays epitopes of said antigen            on its surface; and        -   ii) measuring the level of at least one cytokine released in            said biological sample;    -   b) instructions for comparing the level of said at least one        cytokine in said biological sample with a reference level of        said at least one cytokine,    -   c) instructions for transmitting to a user interface a result of        said comparison, wherein an increase in the level of said at        least one cytokine in said biological sample from the subject as        compared to a reference level indicates a cell mediated immune        response (CMI) to the target antigen in the subject.

In another aspect, provided herein is a system for detecting a pathologyof interest in a subject, the system comprising a computer processor acomputer-readable physical storage medium having instructions recordedthereon sufficient to implement a process, employing the computerprocessor, for measuring a cell-mediated immune response, theinstructions for said process comprising:

-   -   a) instructions for receiving data regarding the level of at        least one cytokine released in a biological sample, said data        obtained by a method comprising the steps of: incubating a        biological sample from said subject with at least one fusion        polypeptide, the fusion polypeptide comprising a portion of an        LF polypeptide lacking LF enzymatic activity but sufficient to        promote transmembrane delivery of said fusion polypeptide to a        cell, said portion fused to a target antigen polypeptide or to a        fragment thereof, wherein the target antigen is expressed in a        tissue affected by the pathology, and wherein said biological        sample comprises cells of the immune system that release at        least one cytokine in response to an antigen; and measuring the        level of at least one cytokine released in said biological        sample; and    -   b) instructions for comparing the level of said cytokine in said        biological sample with a reference level of the same cytokine;        and    -   c) instructions for transmitting to a user interface a result of        the comparison of (b), wherein an increase in the level of said        at least one cytokine in said biological sample from the subject        as compared to a reference level identifies the subject as        having, or having an increased risk of having said pathology.

In another aspect, provided herein is a computer-readable physicalstorage medium having instructions recorded thereon sufficient toimplement a process, employing a computer processor, for measuring acell mediated immune response (CMI) to a target antigen in a subject,the instructions for said process comprising:

-   -   a) instructions for receiving data regarding the level of at        least one cytokine released in a biological sample, said data        obtained by a method comprising the steps of: i) incubating a        biological sample from said subject with at least one fusion        polypeptide, the fusion polypeptide comprising a portion of an        LF polypeptide lacking LF enzymatic activity but sufficient to        promote transmembrane delivery of said fusion polypeptide to a        cell, said portion fused to a target antigen polypeptide or to a        fragment thereof, wherein the target antigen is expressed in a        tissue affected by the pathology, and wherein said biological        sample comprises cells of the immune system that release at        least one cytokine in response to an antigen; and ii) measuring        the level of at least one cytokine released in said biological        sample; and    -   b) instructions for comparing the level of said cytokine in said        biological sample with a reference level of the same cytokine;        and    -   c) instructions for transmitting to a user interface a result of        the comparison of (b), wherein an increase in the level of said        at least one cytokine in said biological sample from the subject        as compared to a reference level identifies the subject as        having, or having an increased risk of having said pathology.

Computer-readable physical storage media useful in various embodimentsinclude any physical computer-readable storage medium, e.g., magneticand optical computer-readable storage media, among others. Carrier wavesand other signal-based storage or transmission media are not includedwithin the scope of physical computer-readable storage media encompassedby the term and useful according to the invention.

A user interface useful in various embodiments includes, for example, adisplay screen or a printer or other means for providing a readout ofthe result of a computer-mediated process. A user interface can alsoinclude, for example, an address in a network or on the world wide webto which the results of a process are transmitted and made accessible toone or more users. For example, the user interface can include agraphical user interface comprising an access element that permits entryof data regarding cytokine release in a biological sample, as well as anaccess element that provides a graphical read out of the results of acomparison transmitted to or made available by a processor followingexecution of the instructions encoded on a computer-readable medium.

I. Compositions and Formulations

In some embodiments, the immunogenic compositions can be useful toelicit a specific antigen immune response against diseases such asinfluenza, and are useful either by inducing mucosal immunity orsystemic immunity, or by a combination of immunity such as humoral,cellular or mucosal. Formulation is as described can be applied tosingle or multiple sites, to single or multiple limbs, or to largesurface areas of the skin by complete immersion.

The LF polypeptides and compositions of the present invention are wellsuited for the preparation of pharmaceutical compositions. Apharmaceutical composition can be administered to any animal which canexperience the beneficial effects of the compositions of the invention.Foremost among such animals are humans, although the invention is notintended to be so limited.

An LF polypeptide such as LFn and a non-linked or non-covalently boundcovalent target antigen can be administered directly to a subject fortreatment (including prophylactic and therapeutic)of a disease thatexpresses the target antigen. For example, in some embodiments, thecompositions as disclosed herein comprising the LFn and at least onenon-linked or non-covalently linked target antigen can be used to elicitan CMI against the target antigen. In some embodiments, the compositionsas disclosed herein comprising an LFn polypeptide and at least onenon-linked or non-covalently linked target antigen can be used for theinhibition of cancer, tumor, or precancerous cells in vivo.Administration is by any of the routes normally used for introducing acompound into ultimate contact with the tissue or cells to be actedupon. The compounds are administered in any suitable manner, preferablywith pharmaceutically acceptable carriers. Suitable methods ofadministering such composition are available and well known to those ofskill in the art, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences, 17thed. 1985)).

Formulations:

The compositions as disclosed herein comprising an LF polypeptide, suchas LFn, and a non-linked or non-covalently linked target antigen can beadministered per se (neat) or in the form of a pharmaceuticallyacceptable salt. When used in medicine, the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltscan conveniently be used to prepare pharmaceutically acceptable saltsthereof and are not excluded from the scope of this invention. Suchpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene-sulfonic,tartaric, citric, methanesulfonic, formic, malonic, succinic,naphthalene-2-sulfonic, and benzenesulphonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts of the carboxylic acidgroup. Thus, the present invention also provides pharmaceuticalcompositions, for medical use, which comprise nucleic acid and/orpolypeptides of the invention together with one or more pharmaceuticallyacceptable carriers thereof and optionally any other therapeuticingredients.

In some embodiments, the compositions comprising an LF polypeptide and atarget antigen as described herein can conveniently be presented in unitdosage form and can be prepared by any of the methods well known in theart of pharmacy. Methods typically include the step of bringing theactive ingredients of the invention into association with a carrierwhich constitutes one or more accessory ingredients.

Preferred compositions suitable for parenteral administrationconveniently comprise a sterile aqueous preparation which is preferablyisotonic with the blood of the recipient. This aqueous preparation canbe formulated according to known methods using those suitable dispersingor wetting agents and suspending agents. The sterile injectablepreparation can also be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent, for example as asolution in 1,3-butane diol. Among the acceptable vehicles and solventsthat can be employed are water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil can be employed including synthetic mono- or diglycerides. Inaddition, fatty acids such as oleic acid find use in the preparation ofinjectables.

Administration:

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intradermal, and subcutaneous routes, includeaqueous and non-aqueous, isotonic sterile injection solutions, which cancontain antioxidants, buffers, bacteriostats, and solutes that renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. The formulations of compounds can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials. Injection solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

In some embodiments, compositions as described herein can beadministered by any means that achieve their intended purpose. Forexample, administration can be by parenteral, subcutaneous, intravenous,intradermal, intramuscular, intraperitoneal, transdermal, or buccalroutes. Alternatively, or concurrently, administration can be by theoral route. The proteins and pharmaceutical compositions can beadministered parenterally by bolus injection or by gradual perfusionover time. Alternatively, a composition can include those suitable fororal, rectal, intravaginal, topical, nasal, ophthalmic or parenteraladministration, all of which can be used as routes of administrationusing the materials of the present invention. Other suitable routes ofadministration include intrathecal administration directly into spinalfluid (CSF), direct injection onto an arterial surface andintraparenchymal injection directly into targeted areas of an organ.Compositions suitable for parenteral administration are preferred. Theterm “parenteral” includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques.Intramuscular administration is preferred.

In some embodiments, compositions comprising an LF polypeptide, such asLFn and a non-linked or non-covalently linked target antigen, eitheralone or in combination with other suitable components (e.g., otheradjuvants and/or carriers) can be made into aerosol formulations (i.e.,they can be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like, ornon-pressurized acceptable propellants such as an spray pump, or similardevices (asthma inhaler) which can be used for oral and/or aerosoladministration of the compositions as disclosed herein.

In alternative embodiments, compositions comprising an LF polypeptideand a target antigen can be formulated to be suitable for oraladministration, and can be presented as discrete units such as capsules,cachets, tablets or lozenges, each containing a predetermined amount ofthe LF polypeptide and a target antigen as discussed herein. In someembodiments, an LF polypeptide and target antigen can be formulated asliposomes or as a suspension in an aqueous liquor or non-aqueous liquidsuch as a syrup, an elixir, or an emulsion.

In some embodiments, the immunogenic compositions can be useful toelicit a specific antigen immune response against diseases such asinfluenza, and are useful either by inducing mucosal immunity or byinducing systemic immunity, or by a combination of immunity such ashumoral, cellular or mucosal.

Preparations which can be administered orally in the form of tablets andcapsules, preparations which can be administered rectally, such assuppositories, and preparations in the form of solutions for injectionor oral introduction, contain about a 0.001 to about 99 percent,preferably from about 0.01 to about 95 percent of active compound(s),together with the excipient.

Suitable excipients are, in particular, fillers such as saccharides, forexample lactose or sucrose, mannitol or sorbitol cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichcan be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, orliquid paraffin. In addition, stabilizers can be added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of one ormore of the active compositions with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the proteins in water-soluble form, for example,water-soluble salts. In addition, suspensions of the proteins asappropriate oil injection suspensions can be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions containing substances whichincrease the viscosity of the suspension include, for example, sodiumcarboxymethyl cellulose, sorbitol, and/or dextran. Optionally, thesuspension can also contain stabilizers.

The proteins are formulated using conventional pharmaceuticallyacceptable parenteral vehicles for administration by injection. Thesevehicles are nontoxic and therapeutic, and a number of formulations areset forth in Remington's Pharmaceutical Sciences, (supra). Nonlimitingexamples of excipients are water, saline, Ringer's solution, dextrosesolution and Hank's balanced salt solution. Formulations according tothe invention can also contain minor amounts of additives such assubstances that maintain isotonicity, physiological pH, and stability.

In some embodiments, a composition comprising an LF polypeptide such asan LFn polypeptide and a non-linked or non-covalently linked targetantigen are formulated in purified form substantially free of aggregatesand other protein materials, preferably at concentrations of about 1.0ng/ml to 100 mg/ml.

Doses:

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular compound employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular compound or vector in a particularpatient

Doses of the pharmaceutical compositions described herein will varydepending on the subject and upon the particular route of administrationused. Generally, for prophylactic vaccination, dosages will range from 1ng to 300 μg of target antigen/LF polypeptide combination. Theproportion of target antigen to LF polypeptide can also vary. E.g., from0.01 target antigen: 1 LF polypeptide (i.e., 100:1 LF to target antigenratio) to 1 target antigen: 0.01 LF polypeptide (e.g., 100:1 targetantigen to LF polypeptide) and all values inbetween. To the extent thatLF polypeptide may form a complex with the target antigen even when notcovalently bound to it, it is contemplated that higher ratios of LFpolypeptide to target antigen may be beneficial. Thus, for example, a100:1 LF polypeptide to target antigen ratio may be preferable to lowerratios, such as 10:1, 1:1 or 0.1:1 of LF polypeptide to target antigenratio.

Preferred doses of the compositions are preferably at least 2 μg/ml. Byway of an example only, an overall dose range of from about, forexample, 1 nanogram to about 300 micrograms might be used for human use.This dose can be delivered at periodic intervals based upon thecomposition. For example on at least two separate occasions, preferablyspaced apart by about 4 weeks. Other compounds might be administereddaily. Pharmaceutical compositions of the present invention can also beadministered to a subject according to a variety of other,well-characterized protocols. For example, certain currently acceptedimmunization regimens can include the following: (i) administrationtimes are a first dose at elected date; a second dose at 1 month afterfirst dose; and a third dose at a subsequent date, e.g., 5 months aftersecond dose. See Product Information, Physician's Desk Reference, MerckSharp & Dohme (1990), at 1442-43. (e.g., Hepatitis B Vaccine-typeprotocol); (ii) for example with other vaccines the recommendedadministration for children is first dose at elected date (at age 6weeks old or older); a second dose at 4-8 weeks after first dose; athird dose at 4-8 weeks after second dose; a fourth dose at 6-12 monthsafter third dose; a fifth dose at age 4-6 years old; and additionalboosters every 10 years after last dose. See Product Information,Physician's Desk Reference, Merck Sharp & Dohme (1990), at 879 (e.g.,Diphtheria, Tetanus and Pertussis-type vaccine protocols). Desired timeintervals for delivery of multiple doses of a particular composition canbe determined by one of ordinary skill in the art employing no more thanroutine experimentation.

The dosage administered will be dependent upon the age, sex, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired. Where vaccination isfor therapeutic purposes, e.g. for treatment of cancer and otherexisting diseases or disorders, the dose ranges for the administrationof the compositions described herein are those large enough to producethe desired effect, whereby, for example, an immune response to theproteins as measured by delayed-type hypersensitivity (DTH), T-cellactivation or antibody production, is achieved, and the disease issubstantially suppressed or treated. The doses should not be so large asto cause adverse side effects, such as unwanted cross reactions,anaphylactic reactions and the like. In some embodiments, a dose forhumans ranges between about 0.001-1 mg/kg body weight. Effective dosesof a composition comprising an LF polypeptide such as an LFn polypeptideand a non-linked or non-covalently linked target antigen for use inpreventing, suppressing, or treating a non-infectious disease can be inthe range of about 1 ng to 100 mg/kg body weight. A preferred dose rangeis between about 10 ng and 10 mg/kg. A more preferred dose range isbetween about 100 ng and 1 mg/kg.

In some embodiments, administration can be accomplished via single ordivided doses, for example, for injectable immunizations that induceimmunity but have potential side effects upon boosting, a transcutaneousboost can be preferable, or oral or nasal immunization can be used to“boost” an immune response against the target antigen. In someembodiments, simultaneous use of injectable and transcutaneousimmunizations could also be used.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity of understandingit will be readily apparent to one of ordinary skill in the art in lightof the teachings herein that certain changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology can be found in Benjamin Lewin, Genes IX,published by Jones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise stated, the present invention can be performed usingstandard procedures, as described, for example in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methodsin Molecular Biology, Elsevier Science Publishing, Inc., New York, USA(1986); Methods in Enzymology: Guide to Molecular Cloning TechniquesVol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., SanDiego, USA (1987)); Current Protocols in Molecular Biology (CPMB) (FredM. Ausubel, et al. ed., John Wiley and Sons, Inc.); Current Protocols inProtein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley andSons, Inc.); Baculovirus Expression Protocols (Methods in MolecularBiology, Vol 39) by Christopher D. Richardson (Editor); Hardcover—450pages Spiral edition (March 1998) Humana Pr; ISBN: 0896032728;Baculovirus Expression Vectors: A Laboratory Manual by David R.O'Reilly, Lois Miller, Verne A. Luckow; Paperback Spiral edition (June1994) Oxford Univ Press; ISBN: 0195091310; The Baculovirus ExpressionSystem : A Laboratory Guide by Linda A. King, R. D. Possee; Hardcover(May 1992) Chapman & Hall; ISBN: 0412371502, which are all incorporatedby reference herein in their entireties.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

In some embodiments of the present invention may be defined in any ofthe following numbered paragraphs:

-   1. A composition for promoting a cell mediated immune (CMI) response    to a target antigen, the composition comprising at least one    isolated target antigen and a portion of a Lethal Factor (LF)    polypeptide lacking LF enzymatic activity, wherein the portion of an    LF polypeptide is not covalently linked to the target antigen, and    wherein the composition does not comprise a protective antigen (PA)    of an exotoxin bipartite protein.-   2. The composition of paragraph 1, wherein said portion of an LF    polypeptide comprises at least the 60 carboxy-terminal amino acids    of SEQ ID NO: 3, or a conservative substitution variant thereof that    promotes a CMI response to the target antigen.-   3. The composition of paragraph 1, wherein said portion of an LF    polypeptide comprises at least the 80 carboxy-terminal amino acids    of SEQ ID NO: 3, or a conservative substitution variant thereof that    promotes a CMI response to the target antigen.-   4. The composition of any one of paragraphs 1 to 3, wherein said    portion of an LF polypeptide comprises at least the 104    carboxy-terminal amino acids of SEQ ID NO: 3, or a conservative    substitution variant thereof that promotes a CMI response to the    target antigen.-   5. The composition of any of paragraphs 1 to 4, wherein the portion    of an LF polypeptide comprises the amino acid sequence corresponding    to SEQ ID NO: 3 or a conservative substitution variant thereof that    promotes a CMI response to the target antigen.-   6. The composition of any one of paragraphs 1 to 5, wherein said    portion of an LF polypeptide does not bind PA polypeptide.-   7. The composition of any one of paragraphs 1 to 6, wherein said    portion of an LF polypeptide substantially lacks amino acids 1-33 of    SEQ ID NO: 3.-   8. The composition of any one of paragraphs 1 to 3, wherein said    portion of an LF polypeptide consists of SEQ ID NO: 4, or a    conservative substitution variant thereof that promotes a CMI    response to the target antigen.-   9. The composition of any one of paragraphs 1 to 3, wherein said    portion of an LF polypeptide consists of SEQ ID NO: 5.-   10. The composition of paragraph 1, wherein the cell is in vivo or    present in an organism.-   11. The composition of paragraph 1, wherein the cell is in vitro.-   12. The composition of paragraph 1 which induces a response by a    cell against a target antigen, when said cell is contacted with the    composition in the presence of the target antigen, and in the    absence of an exotoxin protective antigen (PA).-   13. The composition of paragraph 1, wherein the target antigen is    selected from the group consisting of pathogen antigen, a tumor    antigen or a endogenous misfolded protein.-   14. The composition of paragraph 13, wherein the pathogen antigen is    selected from the group consisting of: Hepatitis A, Hepatitis B,    Hepatitis C, Avian flu virus, ebola virus, west nile virus,    influenza virus, Herpes Simplex Virus 1, Herpes Simplex Virus2,    HIV2, HIV1 and other HIV1 strains.-   15. The composition of paragraph 13 or 14, wherein the pathogen    antigen is not an antigen expressed by B. anthracis.-   16. The composition of paragraph 1, wherein the composition    optionally comprises at least one adjuvant.-   17. The composition of paragraph 16, wherein the adjuvant is    selected from a group comprising of; complete Freud's Adjuvant,    Incomplete Freud's Adjuvant, CM-CSF, QS21, CpG, RIBI Detox, of;    IL-2, Ig-IL-2, B7, ICAM, LFS, dextran, polyvinyl pyrrolidones,    polysaccharides, starches, polyvinyl alcohols, polyacryl amides,    polyethylene glycol(PEG), poly(alkylenes oxides),    monomethoxy-polyethylene glycol polypropylene glycol, block    copolymers of polyethylene glycol, polypropylene glycol.-   18. A method of promotiong a cell-mediated immune response to a    cell, the method comprising contacting said cell with a target    antigen in the presence of a portion of a B. anthracis LF    polypeptide lacking LF enzymatic activity, wherein the portion of    a B. anthracis LF polypeptide lacking LF enzymatic activity is not    covalently linked to the target antigen, and wherein said cell is    not contacted with a protective antigen (PA) of an exotoxin    bipartite protein, whereby a cell-mediated immune response to the    target antigen is promoted.-   19. A composition for delivering a target antigen to a cell, the    composition comprising at least one target antigen and a portion of    a B. anthracis LF polypeptide lacking LF enzymatic activity, wherein    the portion of a B. anthracis LF polypeptide is not covalently    linked to the target antigen, and wherein the composition does not    comprise a protective antigen of B. anthracis exotoxin bipartite    protein.-   20. The composition of paragraph 19, wherein said portion of a B.    anthracis LF polypeptide comprises at least the 60 carboxy-terminal    amino acids of SEQ ID NO: 3, or a conservative substitution variant    thereof that promotes transmembrane delivery.-   21. The composition of paragraph 19, wherein said portion of a B.    anthracis LF polypeptide comprises at least the 80 carboxy-terminal    amino acids of SEQ ID NO: 3, or a conservative substitution variant    thereof that promotes transmembrane delivery.-   22. The composition of any one of paragraphs 19-21, wherein said    portion of a B. anthracis LF polypeptide comprises at least the 104    carboxy-terminal amino acids of SEQ ID NO: 3, or a conservative    substitution variant thereof that promotes transmembrane delivery.-   23. The composition of any of paragraphs 19-22, wherein the portion    of a B. anthracis LF polypeptide comprises the amino acid sequence    corresponding to SEQ ID NO: 3 or a conservative substitution variant    thereof that promotes transmembrane delivery.-   24. The composition of any one of paragraphs 19-23, wherein said    portion of a B. anthracis LF polypeptide does not bind B. anthracis    PA polypeptide.-   25. The composition of any one of paragraphs 19-24, wherein said    portion of a B. anthracis LF polypeptide substantially lacks amino    acids 1-33 of SEQ ID NO: 3.-   26. The composition of any one of paragraphs 19-21, wherein said    portion of a B. anthracis LF polypeptide consists of SEQ ID NO: 4,    or a conservative substitution variant thereof that promotes    transmembrane delivery.-   27. The composition of any one of paragraphs 19-21, wherein said    portion of a B. anthracis LF polypeptide consists of SEQ ID NO: 5.-   28. The composition of paragraph 19, wherein the cell is in vivo or    present in an organism.-   29. The composition of paragraph 19, wherein the cell is in vitro.-   30. The composition of paragraph 19 which induces a response by a    cell against a target antigen, when said cell is contacted with the    composition in the presence of the target antigen, and in the    absence of an exotoxin protective antigen (PA).-   31. The composition of paragraph 19, wherein the target antigen is    selected from the group consisting of pathogen antigen, a tumor    antigen or a endogenous misfolded protein.-   32. The composition of paragraph 31, wherein the pathogen antigen is    selected from the group consisting of: Hepatitis A, Hepatitis B,    Hepatitis C, Avian flu virus, ebola virus, west nile virus,    influenza virus, Herpes Simplex Virus 1, Herpes Simplex Virus2,    HIV2, HIV1 and other HIV1 strains.-   33. The composition of paragraph 31 or 32, wherein the pathogen    antigen is not an antigen expressed by B. anthracis.-   34. The composition of paragraph 19, wherein the composition    optionally comprises at least one adjuvant.-   35. The composition of paragraph 34, wherein the adjuvant is    selected from a group comprising of; complete Freud's Adjuvant,    Incomplete Freud's Adjuvant, CM-CSF, QS21, CpG, RIBI Detox, of;    IL-2, Ig-IL-2, B7, ICAM, LFS, dextran, polyvinyl pyrrolidones,    polysaccharides, starches, polyvinyl alcohols, polyacryl amides,    polyethylene glycol(PEG), poly(alkylenes oxides),    monomethoxy-polyethylene glycol polypropylene glycol, block    copolymers of polyethylene glycol, polypropylene glycol.-   36. A method of delivering a target antigen to the cytosol of a    cell, the method comprising contacting said cell with a target    antigen in the presence of a portion of a B. anthracis LF    polypeptide lacking LF enzymatic activity, wherein the portion of    a B. anthracis LF polypeptide lacking LF enzymatic activity is not    covalently linked to the target antigen, and wherein said cell is    not contacted with a protective antigen (PA) of an exotoxin    bipartite protein, whereby the target antigen is delivered to the    cytosol of the cell.-   37. A method of paragraph 36, wherein the delivery of said target    antigen induces a cell-mediated immune (CMI) response to said target    antigen by said cell.-   38. The method of paragraph 36, wherein said portion of an LF    polypeptide corresponds to SEQ ID NO: 5 or a functional fragment    thereof.-   39. The method of paragraph 36, wherein said portion of a B.    anthracis LF polypeptide comprises at least the 60 carboxy-terminal    amino acids of SEQ ID NO: 3, or a conservative substitution variant    thereof that promotes transmembrane delivery.-   40. The method of paragraph 36, wherein said portion of a B.    anthracis LF polypeptide comprises at least the 80 carboxy-terminal    amino acids of SEQ ID NO: 3, or a conservative substitution variant    thereof that promotes transmembrane delivery.-   41. The method of paragraph 36, wherein said portion of a B.    anthracis LF polypeptide comprises at least the 104 carboxy-terminal    amino acids of SEQ ID NO: 3, or a conservative substitution variant    thereof that promotes transmembrane delivery.-   42. The method of paragraph 36, wherein the portion of a B.    anthracis LF polypeptide comprises the amino acid sequence    corresponding to SEQ ID NO: 3 or a conservative substitution variant    thereof that promotes transmembrane delivery.-   43. The method of paragraph 36, wherein said portion of a B.    anthracis LF polypeptide does not bind B. anthracis PA polypeptide.-   44. The method of paragraph 36, wherein said portion of a B.    anthracis LF polypeptide substantially lacks amino acids 1-33 of SEQ    ID NO: 3.-   45. The method of paragraph 36, wherein said portion of a B.    anthracis LF polypeptide consists of SEQ ID NO: 4, or a conservative    substitution variant thereof that promotes transmembrane delivery.-   46. The method of paragraph 36, wherein said portion of a B.    anthracis LF polypeptide consists of SEQ ID NO: 5.-   47. The method of paragraph 36, wherein the exotoxin is B.    anthracis.-   48. The method of paragraph 36, wherein the cell is in vivo or    present in an organism.-   49. The method of paragraph 36, wherein the cell is in vitro.-   50. The method of paragraph 36, further comprising administering to    the cell at least one other adjuvant, wherein the adjuvant does not    comprise SEQ ID NO: 3 or SEQ ID NO: 4.-   51. The method of paragraph 50, wherein the adjuvant is selected    from a group comprising of; complete Freud's Adjuvant, Incomplete    Freud's Adjuvant, CM-CSF, QS21, CpG, RIBI Detox, of; IL-2, Ig-IL-2,    B7, ICAM, LFS, dextran, polyvinyl pyrrolidones, polysaccharides,    starches, polyvinyl alcohols, polyacryl amides, polyethylene    glycol(PEG), poly(alkylenes oxides), monomethoxy-polyethylene glycol    polypropylene glycol, block copolymers of polyethylene glycol,    polypropylene glycol.-   52. The method of paragraph 36, wherein the target antigen is    selected from the group consisting of pathogen antigen, a tumor    antigen or a endogenous misfolded protein.-   53. The method of paragraph 52, wherein the pathogen antigen is    selected from the group consisting of: Hepatitis A, Hepatitis B,    Hepatitis C, Avian flu virus, ebola virus, west nile virus,    influenza virus, Herpes Simplex Virus 1, Herpes Simplex Virus2,    HIV2, HIV1 and other HIV1 strains.-   54. The method of paragraph 52, wherein the pathogen antigen is not    an antigen expressed by B. anthracis.-   55. The use of a composition of paragraph 1 to induce a cell    mediated response against a target antigen by a cell, wherein the    cell is contacted with the composition in the presence of the target    antigen, and in the absence of an exotoxin protective antigen (PA).-   56. The composition of paragraphs 1 or 19, further comprising at    least one additional immune adjuvant.-   57. The composition of paragraph 56, wherein the immune adjuvant is    selected from the group consisting of; Alum, Complete Freud's    Adjuvant, Incomplete Freud's Adjuvant, CM-CSF, QS21, CpG, RIBI    Detox.-   58. The composition of paragraph 56, wherein the immune adjuvant is    a cytokine selected from the group consisting of; IL-2, Ig-IL-2.-   59. The composition of paragraph 56, wherein the immune adjuvant is    a co-stimulatory molecule selected from the group consisting of; B7,    ICAM, LFS.    The composition of paragraph 56, wherein the immune adjuvant is a    non-antigenic polymeric substance selected from the group consisting    of; dextran, polyvinyl pyrrolidones, polysaccharides, starches,    polyvinyl alcohols, polyacryl amides, polyethylene glycol(PEG),    poly(alkylenes oxides), monomethoxy-polyethylene glycol    polypropylene glycol, block copolymers of polyethylene glycol,    polypropylene glycol.-   61. The method of any of the above paragraphs, wherein SEQ ID NO: 3    or SEQ ID NO: 4 is codon optimized for production in bacterial    cells.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

The contents of all references cited throughout this application, aswell as the figures and tables therein are incorporated herein byreference.

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All references described herein are incorporated herein by reference.

The invention claimed is:
 1. A composition for promoting a cell mediatedimmune (CMI) response to a target antigen, the composition comprising atleast one isolated target antigen and a portion of a Lethal Factor (LF)polypeptide lacking LF enzymatic activity, wherein the portion of an LFpolypeptide consists of SEQ ID NO: 4 or SEQ ID NO: 5, or a conservativesubstitution variant of SEQ ID NO: 4 or SEQ ID NO: 5 that promotes a CMIresponse to the target antigen, wherein the LF polypeptide is notphysically linked to the target antigen, and wherein the compositiondoes not comprise a protective antigen (PA) of an exotoxin bipartiteprotein, and wherein the LF polypeptide is glycosylated, or the targetantigen and LF polypeptide are glycosylated.
 2. The composition of claim1, wherein the cell mediated immune response is in vivo or present in anorganism or in vitro.
 3. The composition of claim 1, wherein the targetantigen is selected from the group consisting of: a pathogen antigen, atumor antigen or an endogenous misfolded protein.
 4. The composition ofclaim 3, wherein the pathogen antigen is selected from the groupconsisting of: Hepatitis A, Hepatitis B, Hepatitis C, Avian flu virus,ebola virus, west nile virus, influenza virus, Herpes Simplex Virus 1,Herpes Simplex Virus2, HIV2, HIV1 and other HIV1 strains.
 5. Thecomposition of claim 1, wherein the composition optionally comprises atleast one adjuvant.
 6. The composition of claim 5, wherein the adjuvantis selected from a group consisting of: Alum; complete Freud's AdjuvantIncomplete Freud's Adjuvant GM-CSF; QS21; CpG; RIBI Detox; IL-2;Ig-IL-2; B7; ICAM; LFS; dextran; polyvinyl pyrrolidones;polysaccharides; starches; polyvinyl alcohols; polyacryl amides;polyethylene glycol(PEG); poly(alkylenes oxides);monomethoxy-polyethylene glycol polypropylene glycol; block copolymersof polyethylene glycol; polypropylene glycol.
 7. A composition fordelivering a target antigen to a cell, the composition comprising twoseparate components wherein at least one of the separate components isglycosylated, a first component comprising at least one target antigenand a second component comprising a portion of a B. anthracis LFpolypeptide lacking LF enzymatic activity and consists of SEQ ID NO: 4or SEQ ID NO: 5, or a conservative substitution variant of SEQ ID NO: 4or SEQ ID NO: 5 that promotes a CMI response to the target antigen,wherein the first component is not physically linked to the secondcomponent, and wherein the composition does not comprise a protectiveantigen of B. anthracis exotoxin bipartite protein.
 8. The compositionof claim 7, wherein the cell is in vivo or present in an organism or invitro.
 9. The composition of claim 7, wherein the target antigen isselected from the group consisting of pathogen antigen, a tumor antigenor a endogenous misfolded protein.
 10. The composition of claim 9,wherein the pathogen antigen is selected from the group consisting of:Hepatitis A, Hepatitis B, Hepatitis C, Avian flu virus, ebola virus,west nile virus, influenza virus, Herpes Simplex Virus 1, Herpes SimplexVirus2, HIV2, HIV1 and other HIV1 strains.
 11. The composition of claim7, wherein the composition optionally comprises at least one adjuvant.12. The composition of claim 11, wherein the adjuvant is selected from agroup consisting of: Alum; complete Freud's Adjuvant; Incomplete Freud'sAdjuvant; GM-CSF; QS21; CpG; RIBI Detox; IL-2; Ig-IL-2; B7; ICAM; LFS;dextran; polyvinyl pyrrolidones; polysaccharides; starches; polyvinylalcohols; polyacryl amides; polyethylene glycol(PEG); poly(alkylenesoxides); monomethoxy-polyethylene glycol polypropylene glycol; blockcopolymers of polyethylene glycol; polypropylene glycol.
 13. Thecomposition method of claim 3, wherein the pathogen antigen is atuberculosis antigen.
 14. The composition method of claim 13, whereinthe tuberculosis antigen is selected from at least one from the groupconsisting of:381; Mtb32A; Mtb16; Mtb72f; Mtb59f; Mtb88f; Mtb71f; Mtb46fand Mtb31f; TbH9 (Mtb 39A); TB1 (CFP); or TB2 (ESAT) or fragmentsthereof.
 15. The composition of claim 1, wherein the LF polypeptide isN-glycosylated or O-glycosylated.
 16. The composition of claim 1,wherein the target antigen is N-glycosylated or O-glycosylated.
 17. Thecomposition of claim 15, where the LF polypeptide is glycosylated on atleast one residue selected from residues 62, 212, 286 of SEQ ID NO:4.18. The composition of claim 7, wherein the LF polypeptide isN-glycosylated or O-glycosylated.
 19. The composition of claim 7,wherein the target antigen is N-glycosylated or O-glycosylated.
 20. Thecomposition of claim 19, where the LF polypeptide is glycosylated on atleast one residue selected from residues 62, 212, 286 of SEQ ID NO:4.